Rapid bioluminescence detection system

专利摘要:
An assay is provided for detecting the activity of a reporter kinase comprising (i) adding said reporter kinase to an assay mixture wherein said reporter kinase is contacted with bioluminescent reagent no more than minutes after being contacted with ADP, and wherein, prior to contacting the reporter kinase with ADP, the assay mixture is substantially free from kinase other than reporter kinase; and (ii) detecting light output from the assay mixture. Methods for detecting the presence of an analyte in a sample and methods for validating a treatment process using the above assay are also provided. Further provided are devices for conducting these assays and methods.
公开号:AU2010204173A1
申请号:U2010204173
申请日:2010-01-07
公开日:2011-07-21
发明作者:Richard J. Hesp；Toryn Poolman；Mark J. Sutton
申请人:Health Protection Agency；
IPC主号:C12Q1-48

专利说明:
WO 2010/079357 PCT/GB2010/050018 RAPID BIOLUMINESCENCE DETECTION SYSTEM The invention relates to the field of rapid bioluminescence detection systems, in particular to rapid and very sensitive bioluminescence detection systems for detecting the activity of reporter kinases. Bioluminescent assays. devices, arid kits for detecting the activity of reporter kinases are also provided. The use of kinases as reporter enzymes has been described in the art. By way of example, the present inventors have described the use of reporter kinases in diagnostic systems for detecting the presence of an analyte in a sample (see WO00/46357), and also in systems for validating the effectiveness of decontamination processes (see W02005/093085). The activity of these reporter kinases is typically detected using an ATP bioluminescence system (e.g. luciferin-luciferase), which generates a light output signal. The light output generated is measured using a luminometer, and these measurements are then correlated with the amount of kinase activity. A potential problem associated with reporter kinase systems is the length of time required to obtain the output signal. To date, the typical time required to obtain an output signal ranges from 30 minutes to several hours. There is thus a need in the art for a quicker and! or simplified reporter system. One or more of the above-mentioned problems is solved by the present invention, which, in a first aspect, provides an assay for detecting the activity of a reporter kinase, comprising: (i) adding said reporter kinase to an assay mixture, wherein said reporter kinase is contacted with ADP, and, no more than 5 minutes after being contacted with ADP, said reporter kinase is contacted with a bioluminescent reagent, WO 2010/079357 PCT/GB2010/050018 wherein, prior to contacting the reporter kinase with ADP. the assay mixture is substantially free from non-reporter kinase (ie. kinase other than reporter kinase); and (ii) detecting light output from the assay mixture. in one embodiment of the invention, the method further comprises the step of recording the light output data obtained in step (ii) on a suitable data carrier. in another embodiment of the invention, the reporter kinase is contacted with the bioluminescent reagent no more than 2 minutes, no more than 1 minute, no more than 30 seconds, or no more than 10 seconds. after being contacted with the ADP. In another embodiment, the reporter kinase is contacted simultaneously with the ADP and the bioluminescent reagent. Thus, there is no significant incubation period (or only a very short incubation period) between contacting the reporter kinase with the ADP and contact with the bioluminescent reagent. The invention can therefore be said to employ a "one-step" bioluminescent detection process. In contrast to the above rapid detection system, conventional reporter systems typically employ a "two-step" detection process: Catalysed by kinase [1] 2ADP , ATP +AMP Catalysed by luciferase [2] ATP + D-luciferin + O . m AMP + PR + oxyluciferin + CO In the first step, the reporter kinases are exposed to a source of ADP substrate, and incubated for a sufficient time to allow the generation of ATP [1]. Then, in a second, separate, step, the luciferin/ luciferase reagent is added to convert the ATP generated WO 2010/079357 PCT/GB2010/050018 3 by the reporter kinase into light [2]. This "two-step" bioluminescent assay has been shown to provide accurate kinase detection. However, its "two-step" nature (iKe. the addition of ADP, incubation, and then separate addition of bioluminescent reagent) has proved cumbersome and slow when detection is carried out "in the field", and not in a laboratory setting. To date, the two reaction steps (illustrated above) have been considered incompatible as AMP generated during step [2] drives the equilibrium of step [1] over to the left-hand side, thereby favoring the re-conversion of ATP generated in step [1] into ADP. Since the light signal output of the system is dependent on the presence of ATP, this makes the detection of kinase activity more difficult. Thus, to date, steps [1] and [2] have been separated either temporally (i.e. by including an incubation step as described above), or spatially (i.e. where the reactions are carried out in separate compartments). Contrary to this dogma, the present inventors have found that reaction steps [1] and [2] can in fact be performed simultaneously, without any significant adverse effect on the sensitivity of the detection of the reporter kinases. The resulting "one-step" bioluminescent assay provides significant advantages in terms of speed and convenience, and is particularly advantageous in point-of-care diagnostic tests, and rapid process release indicators, i.e. for the detection of kinase activity in the field rather than in the laboratory. In addition, in order to ensure a high sensitivity and accuracy of detection, the present inventors have found it advantageous to ensure that, prior to the addition of any ADP, the sample containing the reporter kinase is substantially free from any non-reporter (ie. contaminating) kinase activity, and/ or any endogenous ATP. As will be clear from the reaction schemes above, the presence of either of these contaminants can significantly adversely affect the sensitivity/ accuracy of the detection of kinase activity. By way of example, non-reporter kinases may convert ADP to ATP and thus generate a false (or incre based) light output signal. Thus, it has been found advantageous to treat the sample WO 2010/079357 PCT/GB2010/050018 4 containing the reporter kinase to remove or inactivate any non-reporter kinase and/ or any endogenous ATP. In one embodiment of the invention, non-reporter kinase is removed and/ or inactivated using one or more of the treatment steps described below. In this regard, preferred non reporter kinases that are inactivated or removed in accordance with the present invention are mammalian. fungal and/ or plant kinases (eg. a mammalian, fungal or plant adenylate kinase). These treatments may be used in any number (preferably one or more, or at least two, or at least three) and/ or in any combination. In all cases, however, the treatment leaves the reporter kinase substantially intact (eg. active in terms of kinase activity). Any one or more of the following treatment steps can be applied to any aspect of the invention. In one embodiment, non-reporter kinase is inactivated by exposure to a temperature of between 50 to 120 C for a period of between 1 and 30 minutes, for example 90 C for 10 minutes, 90 C for 3 minutes, 90 C for 1 minute, 120 C for 3 minutes, or 120 C for 1 minute. The temperature and duration of the inactivation process denature non-reporter kinase whilst leaving the activity of the reporter kinase substantially intact. In a further embodiment. non-reporter kinase is removed/ inactivated using a chemical denaturation treatment. Examples of suitable treatments include exposure to a chaotrope such as urea (e.g. concentrations greater than 2M urea) or guanidine (e.g. concentrations greater than 1 M guanidine), exposure to a detergent (e.g. greater than 0.5% SDS, sarlkosyl or triton X-100) exposure to a free-radical generator (e.g. > 1000pprn active chlorine derived from sodium hypochlorite or equivalent reagents) or exposure to an oxidative treatment. In another embodiment, non-reporter kinase is removed/ inactivated using an enzymatic denaturation treatment. Examples of suitable enzymes include highly processive proteases, such as e.g. Prionzyme@, Properase@, proteinase-K, and thermolysin.
WO 2010/079357 PCT/GB2010/050018 5 In a further embodiment, non-reporter kinase is removed/ inactivated by exposure to a selected pH (e.g. below pH 4, or greater than pH 11 using buffers such as 50rnM CAPS pH 11), a selected salt concentration (e.g. >2M ammonium sulphate), EDTA, or combinations thereof. In a further embodiment, non-reporter kinase is rernoved/ inactivated by the addition of an inhibitor, which selectively or specifically inhibits the non-reporter kinase (i.e. the inhibitor inactivates the non-reporter kinase, whilst leaving the activity of the reporter kinase substantially intact). Examples of suitable inhibitors include: staurosporine; vanadate (eg. orthovanadate or decavanadate); glycerophosphate; Diadenosine phosphates such as Ap6A (Diadenosine hexaphosphate), Ap5A (Diadenosine pentaphosphate), Ap4A (Diadenosine tetraphosphate), and! or Ap3A (Diadenosine triphosphate); vitamin C; AMP-PCP; AMP-PNP; AMP-S; ATP-yS; and Ara-ATP. Competitive inhibitors of non-reporter kinases (eg. of non-reporter adenylate kinase) are preferred (eg. Diadenosine phosphate inhibitors such as Ap4A and! or Ap5A). in one embodiment, the inhibitor selectively or specifically inhibits rnammalian and fungal (eg. yeast) and plant non-reporter kinases. In another embodiment, the inhibitor (eg. Ap5A) selectively or specifically inhibits mammalian and fungal (eg. yeast) non-reporter kinases. In a further embodiment, the inhibitor (eg. Ap4A and/ or Ap6A) selectively or specifically inhibits mammalian non-reporter kinases. inhibitors may be determined empirically, for example for different sarnples or matrices. For exarnple a range of different inhibitors have been shown experimentally to provide discrimination between a reporter kinase (e.g. a kinase from S.acidocaldarius, Tmaritirna. or Chlamydia pneumonae) and a non-reporter kinase such as a mammalian tissue-derived kinase as represented by rabbit muscle adenylate kinase (Figure 4 and Figure 7). Thus, in one embodiment, the use of one or more inhibitor such as Ap4A, Ap5A and/ or Ap6A substantially reduces the activity of non-reporter kinase (eg. endogenous tissue-derived kinase such as adenylate kinase) - the employed inhibitor concentrations are typically in the low rnicromolar range and have no significant effect on a reporter kinase. By way of further example, Ap5A discriminates reporter kinase from non-reporter kinase (eg. fungal adenylate kinase) represented here by the enzyme WO 2010/079357 PCT/GB2010/050018 6 from Saccharomyces cerevisiae. On this basis inhibitor selection may be based on both the nature of the reporter kinase and the background (ie. non-reporter kinase) of the sample. Examples of suitable reporter kinase applications of the present invention are illustrated in Table 1 (below) - also shown are examples of contaminating non-reporter kinases typically encountered in said applications. Table I also lists, purely by way of example, a selection of inhibitors that may be employed (eg. by addition to sample preparation buffers) in the context of the present invention. Table I Example of reporter Example of non- Example of Utility kinase reporter kinase inhibitor Bacterial kinase (eg. AK): Marnralan-derived Mammalian Detection of bacterial e.g from Chiamydia tissue, cell or sample kinase inhibitor infection in a patient pneumonia (eg. Ap4A, Ap5A and! or Ap6A) Bacterial kinase (eg. AK): Mammalan-derived Mammalian Detection of viable e.g. frcm Burkodheria tissue, cell or sarnple kinase inhibitor bacterial pathogens in a pseudorna!/e& (eg. Ap4A. Ap5A celI culture model and/ or Ap6A) Archaeal kinase (eg. AK); Mammalian-derived Mamrnalian Detection of an analyte in e.g from S. aciocaiarius tissue, cell or sarnple kinase inhibitor a patient sample (eg. Ap4A. ApSA and/ or Ap6A) Bacterial kinase (eq. AK); Fungal-derived cell or Fungal kinase Detection of bacterial eg from Therrmatoga culture inhibitcr (eg. contaminant in a brewing maritime Ap5A) vessel Fungal kinase (eg. AK): e.g. Mammalian-derived Mammalian Detection of a fungal from S.cerevisiae tissue, cell or sarnple kinase inhibitor contaminant in a tissue (eg. Ap4A and/ culture or Ap6A) Bacterial kinase (eg. AK): Plant-derived tissue, cell Plant kinase Detection of a bacterial e.g. from Pseudomonas or sarnple inhibitor (eqg. contaminant in a plant cell aeruginosa Ap4A and! or culture Ap5A) WO 2010/079357 PCT/GB2010/050018 7 Fungal kinase (eg. AK): e.g. Plant-derived tissue, cell Plant kinase Detection of a fungal from Phytophthora or sample inhibitor (eq. pathogen in a plant ramorum Ap4A and/ or Ap6A) Protozoan kinase (eg. AK); Mammalianderived Mamnahart Detection of a malarial e.g, from Piasmodium tissue. cell or sample kinase inhibitor infection in a patient blood aisciparum (eg. Ap3A and! sample or Ap4A) I another embodiment, non-reporter kinase may be separated from reporter kinase on the basis of size. By way of example, the sample containing the reporter kinase can be run on a filtration device, which separates the non-reporter kinase and the reporter kinase on the basis of size, with the reporter kinase being retained on a suitable filter whilst the non-reporter kinase passes through (see e.g. Example 14, and Figure 6). This may be achieved by coupling the reporter kinase to a particle or within a vesicle which is preferentially retained by the filter. In either case the adherence of the reporter kinase to the filter does not result in the significant loss of the reporter kinase activity. Suitable filter matrices include: nitrocellulose, cellulose acetate or paper filters. Filter matrices typically employ a range of pore sizes, such as from 0,2 pm to 20 pm or larger depending on the nature of the particulate carrier employed. Physical size may also be used as a basis for separation of non-reporter kinase from reporter kinase using gel filtration or size exclusion chromatography. In one embodiment, the reporter kinase has a lower molecular weight than the non-reporter kinase. In another embodiment, the reporter kinase has a higher molecular weight than the non-reporter kinase. By way of example, the reporter kinase may have a molecular weight of at least 40 to 80 kDa, whereas the non-reporter kinase may have a molecular weight of no more than 30 kDa. When run through a size exclusion resin or mernbrane, this provides very efficient separation with the larger protein (eg. the reporter kinase) running at or near the void volume of the matrix (hence running quickly) whilst the non reporter kinase (eg. endogenous kinase such as marrnalian tissue kinase) interacts with the pores of the rnatrix and elutes more slowly. Suitable "higher molecular weight" reporter kinases may be obtained frorn Archael sources (e.g. trimeric adenylate kinases WO 2010/079357 PCT/GB2010/050018 8 enzymes from Aracheal sources), which are in the region of 60kDa in size compared to the 21-22kDa of contaminating non-reporter kinase (eg. endogenous kinase such as marnrnalian tissue kinase). In addition, the size differential between the reporter and non-reporter kinase may be enhanced by the addition of a protein or antibody fragment (e.g. a single chain antibody variable region (scFv), by either chemical conjugation or genetic fusion and recombinant expression) to the reporter kinase. For example, a trimeric adenylate kinase fused to a single chain antibody variable region (scFv) has a size in the order of 120kDa (based on an scFv size of approximately 20kDa, attached to each of the three subunits). In a further embodiment, separation of non-reporter kinase from reporter kinase can be achieved by the use of surface charge. In one embodiment, the isoelectric point of the reporter kinase may be lower than that of the non-reporter kinase. In another embodiment, the isoelectric point of the reporter kinase may be higher than that of the non-reporter kinase. As such, the reporter kinase can be separated from the non reporter kinases with selective binding of either the reporter kinase or the non-reporter kinase to a cation exchange matrix or anion exchange matrix at a suitable pH. The isoelectric point of reporter kinase is frequently in the high basic range; e.g. the tAK from S.acidocaldarius has a predicted pl of 9.03 (although the inventors have demonstrated that the actual pi is in excess of pH10 - see Table 2). By contrast, the majority of non reporter kinases that could interfere with the assay typically have a lower isoelectric point, e.g. a pi in the region of pH7. As such, the reporter kinase can be separated from the non-reporter kinases with selective binding of the reporter kinase, by the use of either a cation exchange resin, membrane or other solid matrix at a pH of at least 8, or using an anion exchange resin, membrane or other solid matrix above pH10. Many of the reporter kinases of the invention retain enzymatic activity in this pH range. Alternatively, non-reporter kinases can be selectively removed by binding them to suitable matrices, e.g. an anion exchange matrix up to pH9. In another embodiment of the invention, non-reporter kinase can be separated from reporter kinase using a "hydrophobic capture" technique. Reporter kinases (eg. those from the Sulfolubus family, and related Sulfolobaceae families such as acidianus, WO 2010/079357 PCT/GB2010/050018 9 metallosphaera, stygiolobus, and sulfurisphaera) show exceptionally tight binding to a variety of surfaces, even when such surfaces are pre-treated or pre-coated (termed "blocked") with other proteins or detergent-based blocking agents. In contrast, the "blocking" of surfaces substantially prevents the binding of non-reporter kinases (eg. mammalian, fungal and/ or plant non-reporter kinases). This difference in physical binding properties allows for an effective separation of reporter kinase from contaminating non-reporter kinases by adherence onto a surface. with the measurement of the reporter kinase being made on that surface after capture. For example, use of a polypropylene of polycarbonate surface) coated with either of the commonly used blocking agents bovine serum albumen (eg. BSA; 3% w/v in neutral buffer) or skimmed milk (eg. 5% w/v in neutral buffer) will completely prevent the binding of non-reporter kinases (eg. endogenous kinases such as mammalian tissue kinases) but not reporter kinase. In this regard, the trimeric reporter kinases such as those derived from S., acidocaldarius, S. soifatarcus and related genera are particularly adherent in these circumstances. One or more of the above treatments for removing/ inactivating non-reporter kinase can be combined to achieve or enhance the desired effect. This may mean that the relative concentrations of one or more of the chemical components may be reduced in the presence of second component. For example, the level of urea required to inactivate non-reporter kinase may be around 2M on its own but can be reduced to 1M in the presence of 0.5 % SDS, as they both exert an effect on the target molecules. Some of the above treatments rnay also have other beneficial effects in clarifying samples being processed and providing greater access to molecules to be detected. In this regard, a preferred application of the present invention is the detection of a microbial infection in a biological sample. Accordingly, the present application provides a sensitive and rapid point-of-care microbial assay. The invention is particularly suited to the rapid detection of bacterial, viral and/ or fungal infections in biological samples, such as the microbial sources listed under 'reporter kinase' in Table 1. Additional microbial infections include those described in the Examples, such as hepatitis species, measles species, norovirus species, legionella species, chlamydia species, listeria species, salmonella WO 2010/079357 PCT/GB2010/050018 10 species, and burkholderia species. The present invention facilitates the detection of microorganisms in stool samples (for example, by the addition of urea and SDS), both in terrns of rnore uniform samples and In the release of the microbial antigens from clumps or aggregates. Similarly, the addition of sodium hypochlorite to a stool sample may simultaneously sterilise the sample (minimising the chance of infections) and reduce the activity of the non-reporter kinase. The precise order/ timing of the steps for removing non-reporter kinase is not critical, provided that these steps are carried out before the reporter kinase comes into contact with ADP. Thus, they can be carried out in the sample preparation phase, or during the assay before the reporter kinase comes into contact with ADP. In one embodiment, the treatment is instead of, or in addition to, a washing step. Adenylate kinase (AK) Structure Mw pI Predicted / origin Actual (if known) S.C idocaldarius Trimer 63330 (3x21110 9,03 / >10 S&sofatavicus Trimer 63975 (3x21325) 8,31 P. furiosus Trimer 7 0 602 (323534) 9.10 A. pemix Triner 70149 (3x23383) 9.31 T.maritima Monomer 26458 6.44 / -6.7 P.abyssi Monomer 26793 8.70 Afulgidus Monomer 24703 5.74 C.trachomatis Monomer 27784 4.63 C.pneumoniae Monomer 23952 7.19 C.difficile Monomer 23700 5.29 B.pseudomallei Monomer 24169 8.03 B.anthracis Monomer 23743 4.80 S.aureus Monomer 23974 4.69 M.tuberculosis Monomer 20124 4.91 A.baumanii Monomer 24022 4.98 R.provazekii Monomer 24501 9.25 Francise!!a tuaensis Monomer 24361 8.06 E coi monomer 23589 5.56 Table 2: Summary of properties of reporter kinases (eg, AKs). As mentioned above, the presence of endogenous ATP may adversely affect the accuracy sensitivity of the assay of the present invention. Thus, in one embodiment, any ATP present prior to addition of ADP is optionally removed using one or more of the treatment steps described below. These treatments may be used in any number (preferably one or more, or at least two, or at least three) and/ or in any combination. In all cases, however, WO 2010/079357 PCT/GB2010/050018 11 the treatment leaves the reporter kinase substantially intact. The treatment steps can be applied to any aspect of the invention. In one embodiment. the removal of endogenous ATP is achieved using an ATPase (e.g. apyrase). The ATPase may then be removed and/ or inactivated before the contact with ADP, to avoid the presence of the ATPase adversely influencing the signal obtained using the reporter kinase. By way of example, an ATPase can be used to remove ATP and then the ATPase is itself destroyed by use of elevated temperature. Alternatively, the ATPase can be immobilised on a device (such as a lateral flow device or filtration device described elsewhere in this specification), such that when ATP flows over the ATPase, the ATP is inactivated. As above, this inactivation step must occur before the reporter kinase comes into contact with the ADP. In a further embodiment, endogenous ATP can be removed by physical means. By way of example, a filtration device can be used, which separates out the ATP on the basis of size in a sirnilar way to that described above for separation of the reporter kinase from non-reporter kinases. Advantageously, the removal of both the ATP and non-reporter kinase can be achieved simultaneously as they are both much smaller than the reporter kinase, either when the latter is on its own or when attached to an antibody, structure or other diagnostic reagent. In another embodiment, endogenous ATP can be removed on the basis of surface charge as described above. The negative charge of the ATP at pH 5.5 allow it to bind to an anion exchange resin, along with non-reporter kinases, but not the reporter kinase. This again effectively separates the contaminating ATP and non-reporter kinase from the signal-generating reporter kinase in a single step. The precise order/ timing of the steps for removing endogenous ATP is not critical, provided that these steps are carried out before the reporter kinase comes into contact with ADP. Thus, they can be carried out in the sample preparation phase, or during the assay before the reporter kinase comes into contact with ADP. In one embodiment, the treatment is instead of, or in addition to, a washing step.
WO 2010/079357 PCT/GB2010/050018 12 Data of the type presented in Figure 3 are helpful when deciding on the type and! or number of background-reduction steps (i.e. removal or inactivation of non-reporter kinase and! or ATP) to use in the assay of a particular sample (although this information does not preclude the use of these steps in any assay type, particularly where infections can influence the background levels of either ATP or reporter kinase). Any suitable kinase enzyme may be used as the reporter kinase in the present invention. In one embodiment, the reporter kinase is an adenylate kinase, acetate kinase or pyruvate kinase, or a combination thereof. The reporter kinase used in the invention may have a trimeric or monomeric structure these tertiary structures are associated with an improved stability of the kinase to conditions such as e.g. temperature, pH, chemical denaturants, or proteases. In one embodiment, the reporter kinase is a non-mammalian, a non-fungal, and/ or a non-plant kinase. In one embodiment, the reporter kinase is a microbial kinase - suitable kinases include Pyrococcus species kinases such as Pvrococcus furiousus kinase, P. abyssi kinase, P furious kinase, P. horikoshii kinase, P. woesii kinase; Sulfolobus species kinases such as Sulfolobus soifataricus kinase. S. acidocaldarius kinase, S. shibatac kinase; Rhodothermus species kinases such as Rhodothermus marines kinase: Thermococcus species kinases such as Thermococcus IitoraIs kinase: Thermotoga species kinases such as Thermatoga maitima kinase, Thermatoga neapoitana kinase; and Methanococcus species kinases such as M. ruber kinase. In another embodiment, the kinase is an Archeoglobus species kinase such as A. fgiius kinase; an Aeropyrum species kinase such as A. pernix kinase; an Aquifex species kinase such as A. pyrophilus kinase, an Alicyclobacillus kinase such as A. acidocaldarius kinase; a Bacillus species kinase such as B. caldotenax BT1 kinase, a Bacillus species PS3 kinase, B. stearothermophil us 11057 kinase, B. stsarothermophilus 12001 kinase, B. thermocatenuLlatus kinase; aclostridial species kinase such as C. stercocorarium kinase; a Thermoanaerobacter species kinase such as T. ethanoiicus kinase, T.
WO 2010/079357 PCT/GB2010/050018 13 thermosulfurogenes kinase, T. celere kinase, T. aquaticus YT1 kinase, T. caldophilus GK24 kinase, T. therrophilus HB8 kinase, In preferred embodiment, the kinase is a T. Itoralis kinase, T. mari/ima kinase, or a T. neapoiltana kinase. In one embodiment, the reporter kinase is thermostable. As well as being resistant to high temperatures, thermostable kinases are also found to be resistant to other biochemical and physical processes that routinely damage or destroy proteins or render them inactive, such as exposure to certain chemicals e.g. chaotropes, free-radical darnage, detergents, extremes of pH, exposure to proteases, protein cross-linking, encapsulation within non-permeable or semi-permeable membranes or polymers, or irreversible immobilisation onto surfaces. (See for example: Daniel RM, Cowan DA, Morgan HW, Curran MP, "A correlation between protein thermostability and resistance to proteolysis", Biochem J. 1982 207:641-4; Rees DC, Robertson AD. "Some thermodynamic implications for the thermostability of proteins", Protein Sci. 2001 10:1187-94: Burdette DS, Tchernajencko V V, Zeikus JG."Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity", Enzyme Microb Technol. 2000 27:11-18; Scandurra R, Consalvi V, Chiaraluce R, Politi L, Engel PC., "Protein thermostability in extremophiles", Biochimie. 1998 Nov;80(11):933-41; and Liao HH., "Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents", Enzyme Microb Technol. 1993 Apr; 1 5(4):286-92, all of which are hereby incorporated by reference in their entirety). In another embodiment, the reporter kinase may be an Ecoi kinase, Clostridium /difficile kinase, Bacillus anthracis kinase, Acinetobacter bauman| kinase, Burkholderia pseudomallei kinase, Chlamydia trachomatis kinase, Charnydia pneumonia kinase, Staphylococcus aureus kinase, Klebsiella pneumonia kinase. Rickettsia prowazekii kinase, Mycobacterium tuberculosis kinase, Saccharomyces cerevisia kinase, Leishmania donovanii kinase, Trypanosoma cruzi kinase, Shigella flexner kinase, Listera monocytogenes kinase, Plasmodiurm falcipa-um kinase, Mycobacterium marinum kinase, Cryptococcus neoformans kinase, Francisela tulraensis kinase, Salmonella spp. kinase, Coxiela bunetii kinase, and/ or Brucela abortus kinase, In WO 2010/079357 PCT/GB2010/050018 14 several of the embodiments. the kinase derived from these organisms is non thermostable, but can be distinguished from non-reporter kinase by the use of different sample treatment, extraction or separation techniques. Many of these reporter kinases, in combination with the method to distinguish their activity from non-reporter kinases, may be used in rapid assays to detect the presence absence, viability or destruction of the organism from which they originate. Such methods are suitable for assessing the presence of an infection within patient sample, tissue or cell population and the effectiveness of different therapeutic regimes or drugs. Examples of specific kinases that have been sequenced and that are suitable for use in the invention are SEQ ID NOs 1-25, 31-36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 61-84. In one embodiment. the kinases used in the invention have at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identity to SEQ ID Nos: 1-25, 31-36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 61-84. Other examples of suitable reporter kinases may be found in W000/46357 and W02005/093085. which are hereby incorporated by reference in their entirety. The stability of the reporter kinases may be increased using a variety of methods well known to those familiar with the art. By way of example, stabilising agents (such as sorbitol up to a concentration of 4M, or other polyols such as ethylene glycol, glycerol, or mannitol at a concentration of up to 2M) may improve the stability of the kinase. Other additives such as xylan, trehalose, gelatin may also provide additional stabilisation effects either individually or in combination. Addition of a range of divalent metal ions, most notably Ca 2 Mg 2' or Mn may also improve stability of the kinase. Chemical modification of the kinases can also be used to improve their stability. Reductive alkylation of surface exposed amino groups by glyoxylic acid (e.g Melik Nubarov (1987) Biotech letts 9:725-730), addition of carbohydrates to the protein surface (e.g. Klibanov (1979) Anal. Biochem. 93:1-25) and amidation (e.g. Klibanov WO 2010/079357 PCT/GB2010/050018 15 (1983) Adv. Apple. Microbiol. 29:1-28) may all increase the stability of the kinase. Further methods including the use of chemical cross-linking agents and the use of various polyrneric supports for enzyme immobilisation are also relevant methods for increasing the stability of enzymes (reviewed in Gupta (1991) Biotech. Apple. Biochem. 14:1-11) Formulation of the kinase in a solution containing up to around 10rng/mi of a suitable carrier protein such as casein or albumin, or the addition of free amino acids such as glycine, tyrosine, tryptophan or dipeptides to the formulation, may increase the stability of the kinase to protease treatments. The genetic modification of enzymes has been shown to provide significant increases in thermal stability and by analogy such mutations are also likely to significantly enhance the stability of the enzymes to other conditions such as protease treatment or gaseous phase sterilisationn". The comparison of the thermostability of the kinase enzymes taken with the defined 3-D structure of the trimeric (archaeal) AKs (Vonrhein et al (1998) J. Mol. Biol. 282:167-179 and Criswell et al (2003) J. Mol. Biol.330:1087-1099) has identified amino acids that influence the stability of the enzyme. Genetically engineered variants of kinases showing improved stability can be generated in a number of ways. Essentially these involve the specific site-directed mutagenesis of amino acids believed to form part of the central core packing region of the trimeric molecule and random "directed evolution" methods where the whole molecule is subjected to subsequent rounds of mutagenesis and selection/ screening of molecules with improved properties. Specific modified enzymes are set out in SEQ ID NOs: 17-19 (several variants are embraced by each reference). These modifications outlined are based on a hybrid approach using a consensus based approach to define regions likely to influence the thermostability of the enzymes based on observed differences between structurally related molecules. This is followed by either defined changes to incorporate the amino acids that correlate with the best thermostability or a random replacement to incorporate every available amino acid at the positions defined as being essential for thermostability.
WO 2010/079357 PCT/GB2010/050018 16 In one embodiment of the invention, the reporter kinases may be bound onto a solid support. Suitable solid supports include a plastic (e.g. polycarbonate, polystyrene or polypropylene) surface, a ceramic surface, a latex surface, a rnagnetic surface, a steel or other rnetallic surface, a flow matrix (as described elsewhere in this specification), a filter membrane, or other polymer surface. The solid support can take the form of e.g. strips, dipsticks, microtitre plates, beads. Binding of the reporter kinase to the solid support may be achieved using any of a wide variety of methods known in the art. In one embodiment, the reporter kinase is bound onto the solid support via standard protein adsorption methods, such as outlined below. Binding of the reporter kinase onto the solid support may be achieved by methods routinely used to link protein to surfaces, e.g. incubation of protein in 0.1M sodium bicarbonate buffer at about pH 9.6 at room temperature for about 1 hour. Alternatively the protein is covalently coupled to the surface using any of a wide range of coupling chemistries known to those familiar with the art. For example an adenylate kinase fusion protein (e.g. to Sup35) derivatised with SPDP (Pierce chemicals; using manufacturer's instructions). reduced with DTT to provide free sulfhydryl groups for cross-linking, is covalently attached to a polystyrene support with a maleimide surface. Plastic surfaces with such sulfhydryl-binding surfaces are well described in the literature. The reporter kinases described in this application have the property that their activity is retained upon derivatisation and cross-linking to such supports. Alternatively an amine reactive surface on a polystyrene or polycarbonate support is used, with a bifunctional cross-linking agent such as monomeric glutaraldehyde, to provide direct non-cleavable cross-linking of the kinase via free amine groups on the protein. UV treatment can also be used to directly link the indicator to a suitable support.
WO 2010/079357 PCT/GB2010/050018 17 Steel surfaces can be treated in a similar way to plastic surfaces to mediate covalent attachment of the kinase. A wide variety of protein cross-linking reagents is available from companies such as Pierce chemical company (Perbio). Reagents reactive to sulfhydryl, amino, hydroxyl and carboxyl groups are designed for coupling proteins but they can equally be used for cross-linking proteins to either naturally reactive or coated solid supports such as plastics, other polymers, glass and metals. Reactive chemistries are also available for cross-linking the enzymes to carbohydrates. For example, the reagents BMPH ((N-[B Maleimidopropionic acid]hydrazide-TFA), KMUH ((N-kMaleimidoundecanoic acid]hydrazide), and MPBH (4-(4-N~Maleimidophenyl)butyric acid hydrazide hydrochloride) can be used to cross link the indicator containing either a free sulfhydryl in the form of a cysteine residue or a chemically derivatised protein reduced to generate a sulfhydryl reactive group, to carbohydrates. This may be particularly important for a solid support which is either a complex carbohydrate (e.g. paper, cellulose-based membranes, gels or resins) or can be coated or treated with a carbohydrate solution to generate a suitably reactive surface. For each type of support the reporter kinase may be formulated in a solution that enhances binding and! or stabilises the bound protein. Such formulations include solutions containing up to 10% (w/v) sucrose, sorbitol, mannitol, cellulose, or polyethylene glycol (PEG). In addition the kinase can be formulated as part of a gel that is applied to the surface or lumen of a suitable support. Examples include alginate, agar or polyacrylamide matrices. In another embodiment, the reporter kinase may be attached to a solid support via a linker that comprises a binding agent specific for an analyte. Details of suitable methods for achieving this attachment are given elsewhere in this specification. The assay described in the first aspect of the invention is particularly suitable for detecting kinase activity in kinase-based analyte detection assays such as those WO 2010/079357 PCT/GB2010/050018 18 described in the applicant's earlier filing, WOOO/46357, the entirety of which is hereby incorporated by reference. Thus, in a second aspect of the invention, there is provided a method for determining the presence of an analyte in a sample, comprising: (i) exposing the sample to a reporter kinase coupled to a binding agent specific for the analyte, so that a cornplex is formed between the reporter kinase and any analyte present in the sample; (ii) separating complexed reporter kinase from uncomplexed reporter kinase; and (iii) measuring the activity of the complexed reporter kinase using an assay according to the first aspect of the invention. The binding agent used in this method (and in any other method described in this specification) is typically an antibody (or a fragment thereof) that binds specifically to the analyte under investigation. The antibody may be obtained using conventional techniques for identification and isolation of specific antibodies, and the assay is thus of application to substantially all analytes against which an antibody can be raised. Alternatively, the binding agent may be selected from the group consisting of lectins, growth factors, DNA/ RNA aptamers, phage or other species that bind specifically to the analyze under investigation. Where a first and second binding agent are involved, these binding agents may be the same or different. The reporter kinase may be coupled to the specific binding agent by conventional techniques. For example, there are numerous ways of labelling immunoreactive biomolecules with enzymes (conjugation). Antibodies, the majority of antigens, and enzymes are all proteins and, therefore, general methods of protein covalent cross-linking can be adapted to the production of immunoassay reagents. The preparation of antibody enzyme conjugates requires mild conditions to ensure the retention of both the immunological properties of the antibody and the catalytic properties of the enzyme. Common methods include, glutaralde hyde coupling, the use of periodate oxidation of glycoproteins to generate dialdehydes capable of forming Schiff-base linkages with free WO 2010/079357 PCT/GB2010/050018 19 amino groups on other protein molecules, and the use of heterobifunctional reagents for example, succinirnidyl-4-(N-maleinidomethyl) cyclohexane-1 -carboxylate (SMCC). In one embodiment of the invention, the above method is a performed as a "capture assay", such as a sandwich assay (sometimes referred to as a two antibody capture assay), an antigen capture assay, or an antibody capture assay. In an example of an antibody capture assay, an analyze is first bound to a solid support, by e.g. non-specific binding. The analyte is then exposed to a reporter kinase linked to a binding agent (e.g. an antibody) specific for the analyte. A complex is thus formed between the analyte and the reporter kinase. Any uncomplexed reporter kinase is removed by one or more routine washing steps. ADP and luciferin / luciferase are then added to the solid support where the ADP is converted to ATP by the reporter kinase complex. The luciferin / luciferase converts the ATP to a light output, which can then be measured and correlated to the amount of analyte present on the solid support. In one embodiment, at any point prior to step (iii), the sample is treated to remove! inactivate non-reporter kinase and! or ATP. Suitable treatments that may be employed in this regard are described earlier in this specification. In one embodiment, the method described in this aspect of the invention is completed within less than 15 minutes, less than 10 minutes, less than 5 minutes, or less than 2 minutes. Example 10 describes the use of a method according this aspect of the invention to detect the presence of Hepatitis C in an oral swab sample. An oral swab sample is taken from the mouth of a patient and dried in an oven at 90 C for 1 minute to remove any non-reporter kinase (eg. endogenous kinase such as mammalian tissue kinase). The swab is then exposed to a conjugate comprising a reporter kinase coupled to an antibody for Hepatitis C antigen. The reporter kinase conjugate forms a complex with any Hepatitis C antigen present on the swab sample. The swab is then rinsed to remove any uncomplexed reporter kinase conjugate, and is inserted into a reagent tube containing ADP and luciferin and luciferase. The reagent tube is transferred to a hand- WO 2010/079357 PCT/GB2010/050018 20 held luminometer and the light output is measured. The light output can then be correlated with the amount of analyte present in the sample. In a third aspect, the invention provides a method for determining the presence of an analyte in a sample, comprising: (i) providing a solid support comprising a reporter kinase, wherein the reporter kinase is attached to the solid support via a linker that comprises a binding agent specific for the analyte; (ii) applying the sample to the solid support., whereby any analyte present in the sample displaces reporter kinase from the solid support; and (iii) measuring the activity of the displaced reporter kinase using an assay according to the first aspect of the invention. In one embodiment, the method described in this aspect of the invention is completed within less than 15 minutes, less than 10 minutes, less than 5 minutes, or less than 2 minutes. By way of example, a clinical sample is provided that is suspected to contain a bacterial toxin. A solid support is also provided, which comprises a reporter kinase linked to the solid support by a binding agent (e.g. an antibody) that is specific for the bacterial toxin. When the sample is applied to the solid support, any bacterial toxin present will competitively interfere with the binding of the antibody to the solid support and will thereby displace the reporter kinase from the solid support. The amount of displaced reporter kinase can then be measured using an assay according to the first aspect of the invention and correlated with the amount of bacterial toxin present in the sample. Example 13 describes the use of this method to detect the presence of norovirus in a clinical sample. In this example, the solid support is coated with an antibody to norovirus (i.e. a binding agent specific for the analyte). A reporter kinase conjugate is formed comprising a reporter kinase conjugated to a VP1 norovirus protein (i.e. the analyte). By virtue of the interaction between the VPi and the antibody, the reporter kinase is attached to the solid support. The clinical sample is then applied to the solid support.
WO 2010/079357 PCT/GB2010/050018 21 Any norovirus (i.e. analyte) present in the sample displaces the reporter kinase conjugate from the solid support. The activity of this displaced reporter kinase is then measured and correlated with the amount of norovirus present in the sample. In one embodiment, the solid support is a flow matrix. The term "flow matrix" is used throughout this specification to mean any liquid-transport solid material that allows for liquid flow therethrough, including materials such as nitrocellulose. nylon, rayon, cellulose, paper, glass fibre, silica, a gel matrix, or any other porous or fibrous materials. In one embodiment, the flow matrix is configured as a substantially planar elongate strip. The flow matrix material can be pre-treated or modified as required. Suitable methods for attaching the reporter kinase to the solid support are described below. The binding agent is as defined above in relation to the second aspect of the invention. 0 An analyte is coupled directly to the surface of the solid support. The reporter kinase is linked to a binding agent specific for the analyte (e.g. an antibody) and thereby associates with the analyte on the surface. The reporter kinase remains attached to the surface until displaced by the presence of either antibody or analyte in the sample. ; An analyte is bound to the solid support via a first binding agent specific for the analyte. The reporter kinase is conjugated to a second binding agent specific for the analyte and thereby associates with the analyte on the surface. The reporter kinase remains attached to the surface (in a sandwich -type arrangement) until displaced by the presence of either antibody or analyte in the sample. * A binding agent specific to the analyte is used to coat the solid support. The reporter kinase is conjugated or genetically fused to the target analyte and thereby associates with the binding agent on the surface. The reporter kinase analyte conjugate is released from the solid support by competing analyte or antibody in the test sample.
WO 2010/079357 PCT/GB2010/050018 The reporter kinase is therefore indirectly attached to the solid support by a linker that comprises a binding agent specific for the analyte. The linker may also comprise the analyte (or a fragment thereof). In one embodiment, at any point prior to step (iii), the sample is treated to remove / inactivate non-reporter kinase and/ or ATP. Suitable treatments are described elsewhere in this specification. in a fourth aspect, the invention provides a method for determining the presence of an analyte in a sample, comprising: (i) providing a solid support on which is attached a first binding agent specific for the analyte; (ii) exposing the solid support to the sample so that any analyte present in the sample becomes attached to the solid support via said first binding agent; (iii) exposing the solid support to a reporter kinase coupled to a second binding agent specific for the analyte, so that the reporter kinase becomes attached to the solid support via the interaction between the second binding agent and the already-bound analyte; (iv) applying the mixture obtained in step (iii) to a filter membrane, wherein the solid support is retained on the filter membrane; and (v) measuring the activity of the retained reporter kinase using an assay according to the first aspect of the invention. In one embodiment, the method described above is completed within less than 15 minutes, less than 10 minutes, less than 5 minutes, or less than 2 minutes. In one embodiment, the solid support is a latex support, or a magnetic support, e.g. a latex bead or a magnetic bead. When the solid support is magnetic, step (iv) may be replaced by exposing the mixture obtained in step (iii) to a magnet, so that the solid support is retained on the magnet.
WO 2010/079357 PCT/GB2010/050018 23 Example 14 describes the use of this method for detecting the presence of legionella in a water sample. Antibodies specific for legionella are attached to a solid support (a latex bead). The latex beads are then exposed to (i) the sample to be tested (potentially containing legionella) and (ii) a reporter kinase coupled to a second antibody specific for legionella. Any legionella present in the sample binds to the antibody on the latex bead. Subsequently. the reporter kinase-antibody conjugate binds to the latex bead via the already-bound legionella. The mixture thus obtained is applied to a filter membrane, which retains the latex beads. The other components of the mixture (e.g. unbound reporter kinase conjugate, ATP, non-reporter kinase (eg. mammalian tissue kinase, plant and/ or fungal kinase endogenous to the test sample etc.) pass through the filter membrane. The reporter kinase retained on the filter membrane is then exposed to ADP and a mixture luciferin / luciferase. and the light output measured using a luminometer. Optionally, the filter membrane can be treated using any of the treatment steps described above for removing any remaining ATP or non-reporter kinase. Suitable filter membranes for use in this aspect of the invention include: nitrocellulose, cellulose acetate or paper filters. Filter matrices typically employ a range of pore sizes from 0.2pm to 20 pm or larger depending on the nature of any particulate carrier used. Example 17 describes the use of this method for detecting the presence of Salmonella in a food sample. The method is essentially as described for Example 14 above, except that a magnetic bead is used as the solid support instead of a latex bead, and the mixture obtained in step (iii) is exposed to a magnet rather than a filter membrane. In one embodiment, at any point prior to step (v), the sample is treated to remove or inactivate non-reporter kinase and! or ATP. Suitable treatments are described elsewhere in this specification. The assay described in the first aspect of the invention is also suitable for detecting kinase activity in kinase-based biological indicator systems such as those described in WO 2010/079357 PCT/GB2010/050018 24 the applicant's earlier filing, W02005/093085, which is hereby incorporated by reference in its entirety. A typical biological indicator is prepared by adsorbing a reporter kinase onto a solid support such as an indicator strip or dipstick. The indicator is then included with a sample (containing a contaminant) to be treated, and the indicator plus sample are subjected to a treatment process. The reduction in activity of the indicator kinase by the treatment is then correlated with the reduction in amount or activity of the contaminant. When a level of activity is determined that is known to correlate with an acceptable reduction in the contaminant, the treatment is then regarded as validated. It has also been found that the performance of these kinase-based indicators can be improved by covalently cross-linking the kinase to a biological component, wherein the biological component is a mimetic/ surrogate of the contaminant. This allows the indicator to more accurately reflect the reaction of the contaminant to the treatment process, which in turn leads to improved indicator accuracy/ sensitivity, and thus fewer "false" process validations. Thus, in a fifth aspect of the invention, there is provided a method of validating a treatment process for reducing the amount or activity of a contaminating biological agent in a sample, comprising the steps of: (i) providing a sample that contains, or is suspected to contain, a contaminating biological agent; (ii) subjecting the sample to a treatment process in the presence of a defined amount of a reporter kinase, wherein the reporter kinase and the contaminating biological agent are both exposed to the treatment process (iii) measuring the residual activity of the reporter kinase using an assay according to the first aspect of the invention; and (iv) comparing said residual activity to a predetermined kinase activity, wherein the pre-determined kinase activity corresponds to a confirmed reduction in the amount or activity of the contaminating biological agent under the same conditions.
WO 2010/079357 PCT/GB2010/050018 25 In one embodiment, steps (i) to (iv) are completed in less than 15 minutes, less than 10 minutes. ess than 5 minutes, less than 2 minutes. In one embodiment, at any point prior to step (iii), the sample is treated to remove/ inactivate non-reporter kinase and! or ATP. Suitable treatments are described elsewhere in this specification. The term "treatment" or "treatment process" encompasses any process that is designed to reduce the amount or activity of a contaminant in a sample. Suitable treatments include one or more of: a selected pH, temperature or pressure, exposing the sample to a protease or other lytic enzyme, exposing the sample to a detergent, a chemical sterilant, radiation, free radicals, or a gas-phase sterilant. In one embodiment, the treatment is designed to reduce the infectious activity (also known as the infectivity) of an infectious biological contaminant, such as TSE. The term "treatment" or "treatment process" also encompasses cleaning and inactivation processes such as high temperature autoclaving with wet or dry steam, ozone sterilisation, H 2 0 2 sterilisation, rendering or other method designed to eliminate or inactivate the contaminant. In one embodiment of the invention, both the reporter kinase and the contaminant are directly exposed to the treatment process, i.e. there is no seal or barrier between the reporter kinase/ contaminant and the treatment process. The reporter kinase and the contaminant are therefore both in direct contact with the treatment process, and are subject to the same treatment conditions, In one embodiment, the contaminating biological agent is selected from the group consisting of bacteria, viruses, spores, toxins, prions, proteins and peptides, In a further embodiment, the reporter kinase is bound onto a solid support using any of the methods described in relation to the first aspect of the invention. In another embodiment of the invention, the reporter kinase is covalently linked to a biological component.
WO 2010/079357 PCT/GB2010/050018 26 The biological component is advantageously a mimetic or surrogate of the contaminant, and therefore reacts to the treatment process in substantially the same way as the contaminant. in one embodiment, the biological component may be the same as, but physically distinct from, the contaminant in the sample that is to be subjected to the treatment process, e.g. if the contaminant is a protein, then the biological component is also a protein; if the contaminant is a blood protein, the biological component is also blood protein: if the contaminant is a DNA molecule, then the biological component is also a DNA molecule; if the contaminant is an RNA molecule then the biological component is also an RNA molecule, etc. for each of the contaminants and biological components disclosed in this specification. Examples of biological components that can be used in the invention include proteins, nucleic acids. carbohydrates and lipids. In one embodiment, the biological component comprises a protein selected from the group consisting of a blood protein, a bacterial protein, a viral protein, a fungal protein, and a self-aggregating or amyloid forming protein. In a further embodiment. the blood protein is selected from the group consisting of blood clotting proteins (e.g. fibrinogen, fibrin peptides, fibrin, transglutaminase substrates, thrombin), serum proteins (e.g. albumin and globulin), platelet proteins, blood cell glycoproteins, and haemoglobin. In another embodiment, the bacterial protein is selected from the group consisting of a bacterial fimbrial protein (e.g CgsA from Ecoi and AgfA from Salmonella), a bacterial toxin protein (e.g. toxins from Bacillus anthracis, Corynebcterium diphtheriae, Clostridium botulinum), a bacterial cell surface protein (e.g. peptidoglycan, lipoproteins), and a bacterial spore protein (e.g. from Gram positive bacteria and having a similar sequence or overall structure to the proteins forming ribbon appendages in Clostridium taeniosporum, chaplin proteins, rodlin proteins).
WO 2010/079357 PCT/GB2010/050018 27 In yet another embodiment, the viral protein is selected from the group consisting of a viral envelope protein, a viral capsid protein, and a viral core protein. Preferably. the viral proteins are frorn a bacteriophage virus (e.g. the MS2 and PP7 proteins), norwalk virus (e.g. capsid protein), rotavirus (e.g. VP2, VP6 and VP7 proteins), coronavirus (e.g. SARS S, E and M proteins), bluetongue virus (e.g. VP2 protein), human papillomavirus (e.g. viral major structural protein, LI), hepatitis B (e.g. small envelope protein HBsAg). Hepatitis C virus (e.g. core, El and E2 proteins), influenza virus (e.g. neuraminidase and haemagglutinin and matrix proteins), poliovirus (e.g. capsid VPO, 1 and 3 proteins), HIV (e.g. Pr55gag, envelope proteins) and dengue B virus (e.g. envelope (e) and pre-rnembrane! membrane (prM/M). In a further embodiment. the fungal protein is selected from the group consisting of hydrophobin proteins (e.g. SC3 from Schizophvllum commune, RodA/B from Aspergillus fumigates, and equivalent proteins from yeast), fungal spore proteins, hyphal proteins, mycotoxins, and fungal prions (e.g. Sup35, Het S, URE 2, Rnqi, New I). In yet a further embodiment, the self-aggregating protein is selected from the group consisting of prions (e.g. PrP. and PrP', Sup35, Het S, Ure 2, Rnq1, New 1), prion mimetic proteins, amyloid fibrils, cell surface adhesins from floc forming and filamentous bacteria in activated sludge, beta amyloid protein, tau protein, polyadenine binding protein, herpes simplex virus glycoprotein B, lung surfactant protein C, CsgA protein from Ecoli, AgfA protein from Salmonella species, bacterial fimbrial proteins, apolipoproteins (e.g. apolipoprotein Al), hydrophobins from fungal species (e.g. SC3 from Schizophylum commune, RodA/B from Aspergillus [umigates), chaplins (e.g. Clips A-H from streptomyces spp), rodlins (e.g. Rd1A and Rd1B from streptomyces spp), gram positive spore coat proteins (e.g. P29a, P29b, GP85 and a SpoVM analogue), and barnacle cement-like proteins (e.g. the 19kDa protein from Balanus albicostatus, and the 20kDa protein from Me gab-lanus rosa, and the novel calcite-dependent cement-like protein from Balanus a/bicostatus). In a further embodiment, the nucleic acid is selected from a DNA molecule and an RNA molecule. Preferably, the nucleic acid is derived from neurological tissue.
WO 2010/079357 PCT/GB2010/050018 28 In a further embodiment, the carbohydrate is selected from the group consisting of exopolysaccharide, lipopolysaccharide (EPS/LPS, sometimes known as endotoxin) (e.g. from Legionella, E coli Staphyiococcus species, Streptococcus species, Pseudomonas species, Acinetobactor specIes. Camp ylobactor species. and Bacillus species), peptidoglycan, cell wall components of plants, fungi and yeast (e.g. chitin, lignin, glucan), rnucin preparations, glycolipids (especially brain derived glycolipids), glycoproteins (e.g. cell surface glycoproteins, Eap1p), spore extracts (e.g. from Bacilus spo, Clostridial spp and other spore-formers), polysaccharides from yeast capsules, and invertebrate secretions (e.g. from molluscan gels). in another embodiment, the lipid is selected from the group consisting of glycolipids (e.g. brain-derived glycolipids), gangliosides (e.g. neuronal cell gangliosides such as GT,, GTI, and gangliosides of more general cell origin such as GM 1 ), and plant oils and lipids. Advantageously, the biological component is part of a biological matrix. The biological matrix rnay be a mimetic of the sample that is to be treated. In one embodiment, the biological matrix comprises one or more components selected from the group consisting of proteins, lipids, nucleic acids, and carbohydrates. or fragments or derivatives thereof. In another embodiment, the biological mnatrix may comprise a mixture of proteins. In a further embodiment, the biological matrix may cornpise one or more components selected from the group consisting of blood, serum, albumin, mucus, egg, neurological tissue, food, culled animal material, and a commercially available test soil. In a further embodiment of the invention, the biological matrix comprises one or more components selected from the group consisting of fibrinogen, thrombin, factor Vill, Ca0, and, optionally, albumin and/ or haemoglobin. Examples of reporter kinases linked to biological components are described in SEQ ID NOs: 34-38, 40, 42, 48, 50, 52, 54, 61, 67, 72, and 73. The biological indicator may be prepared by covalently linking a reporter kinase to an appropriate biological component. Any suitable method of covalent attachment known in WO 2010/079357 PCT/GB2010/050018 29 the art may be used. in one embodiment, the kinase is genetically or chemically cross-linked to the biological component. Chemical cross-linking may be achieved using a range of homo- and hetero-bifunctional reagents commonly used for cross-linking of proteins for the generation of enzyme conjugates or other related purposes. For example, in an indicator comprising fibrin as the biological component, the fibrin and the reporter kinase may be derivatised with the addition of SPDP (Perbio) to primary amine groups. The reporter kinase can then be reduced to generate a reactive thiol group and this is then mixed with the fibrin to produce covalent fibrin- kinase linkages. The reporter kinases can also be chemically crossilinked to carbohydrates, lipids or other glycoconjugates using heterobifunctional agents following treatment of the target carbohydrate with meta-periodate. Alternatively, the indicator may be prepared as a fusion protein. This is achieved by fusing a synthetic gene encoding an appropriate kinase (e.g. the gene encoding AK from Sulfolobus acidocadarius or Thermatoga neopolitana) to a gene encoding an appropriate biological component. Methods according to this aspect of the invention are illustrated in Examples 18-21. In a sixth aspect of the invention, there is provided a device for detecting the activity of a reporter kinase in a sample, comprising: an elongate flow matrix, wherein said flow matrix comprises: (i) a sample-receiving zone: and (ii) a detection zone, located downstream of the sample-receiving zone, comprising a mixture of ADP and a bioluminescent reagent; wherein, in use, a sample is applied to the sample-receiving zone and is drawn along the flow matrix to the detection zone.
WO 2010/079357 PCT/GB2010/050018 30 In use. the sample is applied to the sample-receiving zone of the device and is allowed to migrate to the detection zone where it comes into contact with the mixture of ADP and bioluminescent reagent. Here, any reporter kinase present in the sarnple acts on the ADP to generate ATP, which in turn reacts with the bioluminescent reagent to produce light. The light output from the detection zone can be readily measured using a luminometer, preferably a hand-held luminometer. In one embodiment. the detection zone of the device is snapped off and placed in a luminometer. The arnount of light produced can then be correlated with the amount of reporter kinase activity. In one embodiment, the device comprises a backing strip on which the elongate flow matrix is positioned. The backing strip may be made from any suitable non-absorbing material, such as a plastic-adhesive backing card. In another embodiment, the flow matrix is at least partially sandwiched between a top and a bottom laminate. The top laminate may include a sample-application window, which provides access to the sample-receiving zone of the flow matrix, and may also include a detection window, which provides access to the detection zone of the flow matrix. The laminates may be made from any suitable non-absorbing material, e.g. a transparent or translucent adhesive plastic film, in one embodiment, the device is a lateral flow device. Lateral flow devices and methods for their construction are well known in the art, being best known as the standard pregnancy test kit. In a further embodiment, the device may comprise a background-reduction zone, situated between the sample-receiving zone and the detection zone. This zone functions to remove/ inactivate any non-reporter kinase and/ or ATP that may be present in the sample before the sample reaches the detection zone. Thus, these contaminants are prevented from interfering with the sensitivity or accuracy of the assay. In one embodiment, the background-reduction removal zone comprises a substance that selectively (or specifically) inhibits non-reporter kinase, whilst leaving the reporter kinase substantially unaffected. Suitable inhibitors are described elsewhere in this WO 2010/079357 PCT/GB2010/050018 31 specification. In another embodiment, the background-reduction zone comprises a protease that selectively destroys non-reporter kinase, whilst leaving the reporter kinase substantially unaffected. Suitable proteases are described elsewhere in this specification. In a further embodiment, the background-reduction zone may be arranged so as to physically capture out non-reporter kinases on the basis of their size, charge, or binding properties as described elsewhere in this specification. The captured non reporter kinases are thus prevented frorn reaching the detection zone. In another embodiment, the background-reduction zone comprises an imrnobilised ATPase, e.g. apyrase. In another embodiment, the background-reduction zone may be arranged so as to physically capture out ATP on the basis of its size or charge as described elsewhere in this specification. The captured ATP is thus prevented from reaching the detection zone. In one embodiment, the ADP in the detection zone of the device is high purity ADP, and the bioluminescent reagent is a mixture of luciferin and luciferase. In another embodiment, the ADP and luciferin/ luciferase are immobilised in the detection zone using conventional immobilisation methods. In a further embodiment. the device is portable. In a further embodiment, the detection zone may include a cationic membrane that retains and concentrates the reporter kinase conjugate for enhanced detection. In another embodiment, the sample-receiving zone may include a suitable dye which also migrates to the detection zone, acting as a control for the proper flow of the sample through the device. This positive internal control may also exploit the use of a cation binding membrane within the detection zone to help retain the dye to provide a clear visual signal. In a seventh aspect of the invention, there is provided a lateral flow device for use in an assay for detecting the presence of an analyte in a sample, comprising: WO 2010/079357 PCT/GB2010/050018 32 a backing strip on which is positioned an elongate flow matrix. wherein said flow matrix comprises: (i) a sample-receiving zone comprising a reporter kinase attached to the flow matrix via a linker comprising a binding agent specific for the analyte; and (ii) a detection zone, located downstream of the sample-receiving zone; wherein, in use, a sample is applied to the sarnple-receiving zone and any analyte present in the sample displaces the reporter kinase from the flow matrix and thereby allows the reporter kinase to migrate to the detection zone. In use, the sample is applied to the sample-receiving zone, and any analyte present in the sample displaces the reporter kinase attached to the sample-receiving zone. Any reporter kinase that is not displaced remains attached to the sample-receiving zone, and this is the case for a sample negative for the presence of the analyte. Thus, only the displaced reporter kinase proceeds to the detection zone where it can be detected and correlated with the amount of analyte present in the sample. The backing strip of the device may be made from any suitable non-absorbing material, such as a plastic-adhesive backing card. in one embodiment. the flow matrix is at least partially sandwiched between a top and a bottom larninate. The top laminate may include a sample-application window, which provides access to the sample-receiving zone of the flow matrix, and may also include a detection window, which provides access to the detection zone of the flow matrix. The laminates may be made from any suitable non-absorbing material, e.g. a transparent or translucent adhesive plastic film. In a further embodiment, the detection zone comprises a mixture of ADP and a bioluminescent reagent. The reporter kinase is attached to the flow matrix by a linker comprising a binding agent specific for the analyte. Binding agents and methods for attaching the reporter kinase to the flow matrix are as described in relation to the second aspect of the invention.
WO 2010/079357 PCT/GB2010/050018 33 In one embodiment, the device may further cornprise a background-reduction zone, situated between the sample-receiving zone and the detection zone. This zone functions to remove/ inactivate any non-reporter kinase and/ or ATP that may be present in the sample before the sample reaches the detection zone. Thus, these contaminants are prevented from interfering with the sensitivity or accuracy of the assay. In one embodiment, the background-reduction removal zone comprises a substance that selectively (or specifically) inhibits non-reporter kinase, whilst leaving the reporter kinase substantially unaffected. Suitable inhibitors are described elsewhere in this specification. In another embodiment, the background-reduction removal zone comprises a protease that selectively destroys non-reporter kinase, whilst leaving the reporter kinase substantially unaffected. Suitable proteases are described elsewhere in this specification. In a further embodiment, the background-reduction zone may be arranged so as to physically capture out non-reporter kinases on the basis of their size, charge, or binding properties as described elsewhere in this specification. The captured non-reporter kinases are thus prevented from reaching the detection zone. In another embodiment, the background-reduction zone comprises an immobilised ATPase, e.g. apyrase. in another embodiment, the background-reduction zone may be arranged so as to physically capture out ATP on the basis of its size or charge as described elsewhere in this specification. The captured ATP is thus prevented from reaching the detection zone. In one embodiment, the ADP in the detection zone of the device is high purity ADP, and the bioluminescent reagent is a mixture of luciferin and luciferase. In another embodiment, the ADP and luciferin/ luciferase are immobilised in the detection zone using conventional immobilisation methods. In another embodiment, the device is portable. In a further embodiment, the detection zone may include a cationic membrane that retains and concentrates the reporter kinase conjugate for enhanced detection.
WO 2010/079357 PCT/GB2010/050018 34 In another embodiment. the sample-receiving zone may include a suitable dye which also migrates to the detection zone, acting as a control for the proper flow of the sample through the device. This positive internal control may also exploit the use of a cation binding membrane within the detection zone to help retain the dye to provide a clear visual signal. in an eighth aspect, the invention provides a method for detecting the activity of a reporter kinase in a sample, wherein the method is conducted using a device according to the sixth aspect of the invention, comprising the steps of: (i) applying the sample to the sample-receiving zone of the device: (ii) allowing the sample to flow through to the detection zone of the device and (iii) detecting the light output from the detection zone. In one embodiment. after step (i), the method further comprises allowing the sample to flow through a background-reduction zone as described in relation to the sixth aspect of the invention. In another embodiment, step (iii) is carried out by snapping off the detection zone of the device, and then placing the detection zone into a luminorneter. In a further embodiment, the method comprises the step of recording the light output data obtained on a suitable data carrier. In a ninth aspect of the invention there is provided a method for detecting the presence of an analyte in a sample using the device described in relation to the seventh aspect of the invention comprising: (i) applying the sample to the sample-receiving zone of the device; (ii) allowing any reporter kinase displaced from the sample-receiving zone to migrate to the detection zone; and (iii) detecting the light output from the detection zone.
WO 2010/079357 PCT/GB2010/050018 35 In one embodiment, after step (i), the method further comprises allowing the sample to flow through a backgroundireduction zone described in relation to the seventh aspect of the invention. In another embodiment, step (iii) is carried out by snapping off the detection zone of the device, exposing the detection zone to ADP and a bioluminescent reagent, wherein the detection zone is exposed to the bioluminescent reagent no more than 5 minutes (or no more than 2 minutes. 1 minute, 30 seconds, or 10 seconds) after having been exposed to the ADP, and then placing the detection zone into a luminometer. In one embodiment, the detection zone is exposed to the ADP and bioluminescent reagent simultaneously. In a further embodiment, the method comprises the step of recording the light output data obtained on a suitable data carrier. In a tenth aspect, the invention provides a kit comprising a device according to the sixth or seventh aspect of the invention, and a luminometer. In one embodiment, the luminometer is a hand-held (i.e. portable) luminometer. DEFINITIONS SECTION The term "light output" means the light that is emitted by the reaction of ATP with the bioluminescent reagent. This light output can be detected using entirely conventional technology, such as a standard luminometer (e.g. a Berthold Orion 96-well microplate luminometer, or a hand-held luminometer). The term "flow matrix" refers to any liquid-transport solid material that allows for liquid flow therethrough, and includes materials such as nitrocellulose, nylon, rayon, cellulose, paper, glass fibre, silica, gel matrices, or any other porous or fibrous materials. in one embodiment, the flow matrix is configured as a substantially planar elongate strip. The flow matrix material can be pre-treated or modified as required. The term "reporter kinase" refers to a kinase enzyme that is not a mammalian, plant and/ or fungal kinase. Thus, in the context of a biological sample to be tested, a reporter WO 2010/079357 PCT/GB2010/050018 36 kinase is a kinase that is not normally present (to any significant degree) in a sample taken from a healthy individual. Put another way, a reporter kinase of the present invention is a kinase that is not normally inherent or endogenous (to any significant degree) in a sample taken from a healthy individual. Reporter kinase may be added to the sample as a separate (ie. exogenous) reagent, e.g as an isolated kinase. Reporter kinases are preferably thermostable. The term "non-reporter kinase" refers to kinase enzyme that is not a reporter kinase as defined above. Non-reporter kinases may also be referred to as endogenous kinases, contaminating kinases, or background kinases. Non-reporter kinases are typically present in a sample taken from a healthy individual. Non-reporter kinase activity can also be defined as activity that is not associated with the reporter kinase. Many non reporter kinases are derived from mesophilic organisms, i.e. organisms that grow best at moderate temperatures (e.g. 25-40 C). Examples of non-reporter kinases include mammalian, plant and/ or fungal kinases - in particular, any of the range of 7 human adenylate kinase isoforms found in varying amounts in clinical sarnples, equivalent proteins in animal species or food derived from them, or kinases (e.g. adenylate kinases) from common commensal organisms in humans or animals. The term "thermostable kinase" refers to a kinase that retains activity after exposure to heat, i.e. that is relatively unaffected by high temperatures. Preferred thermostable kinases retain at least 70% activity (or 80% activity, 90% activity, 95% activity, or 1 00% activity.) after exposure to a temperature of between 50-120 C. Particularly preferred thermostable kinases retain at least 70% activity (or 80% activity, 90% activity, 95% activity, or 100% activity) after exposure to 50 C for 30 minutes, or after exposure to 60 C for 30 minutes, or after exposure to 70 C for 30 minutes, or after exposure to 80 C for 20 minutes, or after exposure to 90 C for 3 minutes, or after exposure to 120 C for 3 minutes. Thermostable kinases may also be more resistant than non-thermostable kinases to a range of other biochemical and physical processes that routinely damage or destroy proteins or render them inactive, such as exposure to certain chemicals e.g. chaotropes, free-radical damage, detergents, extremes of pH, exposure to proteases, protein cross-linking, encapsulation within non-permeable or semi-permeable WO 2010/079357 PCT/GB2010/050018 37 membranes or polymers, or irreversible irnrnobilisation onto surfaces. In a particular embodiment, thermostable kinases may retain at least 70% activity (or 80% activity, 90% activity, 95% activity, or 100% activity) after exposure to one or more of the biochemical and physical processes described above. In all cases. this "retained activity" can be readily confirmed using conventional tests, in brief, the kinase is incubated with ADP under the given treatment conditions for a given amount of time, and then analysed for residual activity by detecting the generation of ATP using luciferin/luciferase and a luminometer. From this, the % of kinase activity retained after the treatment can be determined. The terms "kinase" and "kinase activity" are used interchangeably throughout this specification. The term "sample" encompasses any item, instrument, surface, fluid or material. Examples include, but are not limited to clinical samples (such as whole blood, serum, oral samples such as saliva, pus, vaginal samples, stool samples, vomitus), environmental samples (such a water, soil, air samples), surgical and medical instruments, microtitre plates, dipsticks, lateral flow devices, hospital gowns, bedclothes, bulk liquids, culled animal material, pharmaceuticals, workbenches, walls and floors, biological matrices, and biological indicators. The terms "substantially free from non-reporter kinase", "free from non-reporter kinase", "substantially free from kinase other than reporter kinase", and "free from kinase other than reporter kinase" are considered synonymous, and are used interchangably throughout the specification to mean that the level of non-reporter kinase is sufficiently low or absent and does not interfere to any significant degree with the sensitivity or accuracy of the assay. In terms of assay read-out, the impact of the non-reporter kinase is usually defined in terms of the signal-to-noise ratio. As such, the term "substantially free" can also be defined as meaning that the non-reporter kinase does not account for more than 10% (preferably not more than 5% or 2%) of the total kinase signal at the limit of detection of the assay.
WO 2010/079357 PCT/GB2010/050018 38 The terms "substantially free from ATP" and "free from ATP" are considered synonymous and are used interchangably throughout the specification to mean that the level of endogenous ATP is sufficiently low or absent and does not interfere to any significant degree with the sensitivity or accuracy of the assay. Endogenous ATP may have an impact on the assay in terms of signal: noise - thus, the "substantially free" term means that any endogenous ATP accounts for not more than 10% (preferably not more than 5% or 2%) of the total signal at the limit of detection of the assay. The term "simultaneously" means at the same time. In the context of the first aspect of the invention where. in one embodiment, the reporter kinase is contacted with ADP and bioluminesecent reagent simultaneously, this means that there is no (or substantially no) separate incubation period between contacting the kinase with ADP and contacting the kinase with the bioluminescent reagent. The term "bioluminescent reagent" refers to any substance or mixture of substances able to react with ATP to generate light. A preferred reagent is a mixture of luciferin and luciferase. The term "RLU" means Relative Light Unit. Relative Light Units are a relative, not absolute, measurement. The figures given in the specification relate to measurements taken using a Berthold Orion 96-well microplate luminometer with injector system using a "flash" method of light measurement for 2 seconds immediately after the addition of the luciferase/luciferin reagents (technical specification photomuliplier measuring light emitted at a wavelength of 300-650nm). To address this issue, manufacturers have generated data for RLU "factors", which allow the data generated by a given luminometer to be normalised to a calibrated standard. Thus, comparisons can be made between different instruments. The RLU factor for the Berthold Orion 96-well microplate luminometer is 1. Accordingly, the RLU values given in the specification can be regarded as standardised/normaflised RLU values. in terms of absolute values, an RLU value can be related to the concentration of ATP required to give said value with the reagents as described in the method. As an WO 2010/079357 PCT/GB2010/050018 39 approximate conversion, and given the linear relationship between RLU values and ATP concentration, the following values can be used: RLU Approximate concentration of ATP / pM 12,000.000 1000 1,200,000 100 120.000 10 12,000 1 1,200 0.1 120 0.01 All references cited in this application are hereby incorporated by reference in their entirety. SEQ ID NOs SEQ ID I Protein sequence of Adenylate kinase frorn Sulfolobus solfa5aricus SEQ ID 2 Protein sequence of Adenylate kinase frorn Sulfolobus acidocaldaius SEQ ID 3 Protein sequence of Adenylate kinase frorn Sulfolobus tokoda/i SEQ ID 4 Protein sequence of Adenylate kinase frorn Pyrococcus furiosus SEQ ID 5 Protein sequence of Adenylate kinase frorn PyrOcoccus horikoshii SEQ ID 6 Protein sequence of Adenylate kinase frorn PyrOcoccus abyssi SEQ ID 7 Protein sequence of Adenylate kinase frorn Mcthanococcus thermnolithotrophicus SEQ ID 8 Protein sequence of Adenylate kinase frorn Mvethanococcus voltae SEQ ID 9 Protein sequence of Adenylate kinase frorn WO 2010/079357 PCT/GB2010/050018 40 Methanococcus jannaschi SEQ ID 10 Protein sequence of Adenylate kinase from Melhanopyrus kandleri SEQ ID 11 Protein sequence of Adenylate kinase from Methanotorris tgneus SEQ ID 12 Protein sequence of Adenylate kinase from Pyroba culum aerophilum SEQ ID 13 Protein sequence of Adenylate kinase from Thermotoga maritira SEQ ID 14 Protein sequence of Adenylate kinase from Aempyrum pemix SEQ ID 15 Protein sequence of Adenylate kinase from Archaeoglobus fulgidus SEQ ID 16 Protein sequence of Adenylate kinase from Pyrococcus abyssi (monomeric adenylate kinase (AdkE)) SEQ ID 17 Protein sequence of Adenylate kinase from Pyrococcus furiosus genetically engineered to provide improved stability SEQ ID 18 Protein sequence of Adenylate kinase from Pyrococcus horikoshii genetically engineered to provide improved stability SEQ ID 19 Protein sequence of Adenylate kinase from Sulifolobus acidocaldarius genetically engineered to provide improved stability SEQ ID 20 Protein sequence of Acetate kinase from Thermatoga maritima SEQ ID 21 Protein sequence of Pyruvate kinase from PyrOcoccus horikoshii SEQ ID 22 Protein sequence of Pyruvate kinase from Suifolobus solfataricus SEQ ID 23 Protein sequence of Pyruvate kinase from Thermotoga maritime SEQ ID 24 Protein sequence of Pyruvate kinase from Pyrococcus furiosus SEQ ID 25 Protein sequence of Acetate kinase from Methanosarcina thermophila SEQ ID 26 DNA sequence encoding the Adenylate kinase from Sulfolobus acidocaldarius SEQ ID 27 DNA sequence encoding the Adenylate kinase from Suifolobus acidocaldarius. wherein codon usage has been optimised for expression of the gene in B-coli WO 2010/079357 PCT/GB2010/050018 41 SEQ ID 28 DNA sequence encoding the Adenylate kinase from Thermotoga maritime SEQ ID 29 DNA sequence encoding the Adenylate kinase from, Thermologa maritira, wherein codon usage has been optimised for expression of the gene in E-coli. SEQ ID 30 DNA sequence encoding the Adenylate kinase from Arhaeoglobus fulgidus. wherein codon usage has been optimised for expression of the gene in E-coli. SEQ ID 31 Protein sequence of Adenylate kinase from Sulfolobus acidocaldaius. wherein codon usage has been optimised for expression of the gene in E-coli (SEQ ID 27). SEQ ID 32 Protein sequence of Adenylate kinase from Thermotocga maritime. wherein codon usage has been optimised for expression of the gene in E-coli (SEQ ID 29). SEQ ID 33 Protein sequence of transglutaminase substrate SEQ ID 34 Protein sequence of Adenylate Kinase from Sulfolobus acidocaldarus fused at the N-terminus with a transgluta miinase (Factor XI1) substrate sequence SEQ ID 35 Protein sequence of Adenylate Kinase from Sulfolobus acidcaldarius fused at the C-terninus with a transglutaminase (Factor X1Il ) substrate sequence SEQ ID 36 Protein sequence of Adenylate Kinase from Sulfolobus aidocaldarius fused at the N-terminus and C-terminus with a transglutaminase (Factor XIl) substrate sequence SEQ ID 37 DNA sequence of transglutaminase (Factor X111) substrate sequence fused to the 5' end of Adenylate Kinase from Thermotoga maritima. SEQ ID 38 Protein sequence of Adenylate Kinase from Thermotoga maritima fused at the N-terminal with a transglutaminase (Factor XIl) substrate sequence.
WO 2010/079357 PCT/GB2010/050018 42 SEQ ID 39 DNA sequence of transglutaminase (Factor XWI) substrate sequence fused to the 3' end of Adenylate Kinase from Thermotoga maritima. SEQ ID 40 Protein sequence of Adenylate Kinase from Thermotoga maritime fused at the C-terminal with a transglutaminase (Factor X111) substrate sequence. SEQ ID 41 DNA sequence of transgilutaminase (Factor XWI) substrate sequence fused to both the 5' and 3' ends of Adenylate Kinase from Thermotoga maritira. SEQ ID 42 Protein sequence of Adenylate Kinase from Themotoga maritime fused at the N- and C-terminal with a transglutarninase (Factor XW) substrate sequence. SEQ ID 43 DNA sequence of complete Sup35 gene construct from Saccharomnyces cerevisiae SEQ ID 44 Protein sequence of complete Sup35 from Saccharomyces cerevsia9& SEQ ID 45 DNA sequence of sup35N (N-terminal domain) codon-biased for optimal expression in E. coli SEQ ID 46 Protein sequence of sup35N (N-terminal domain) SEQ ID 47 DNA sequence of E-coli codon biased Adenylate Kinase from Sulfolobus acidcalarius fused at the N-terminus with Sup35 N terminal domain from Saccha-omyces cervisiae SEQ ID 48 Protein sequence of Adenylate Kinase from Sulfolobus acidcaldarius fused at the N-terminus with Sup35 Nterminal domain from Saccharomyces cervisiae SEQ ID 49 DNA sequence of E. coli codon biased Adenylate Kinase from Sulfolobus acidcaldarius fused at the C-terminus with Sup35 N terminal domain from Saccha-omyces cervisiae SEQ ID 50 Protein sequence of Adenylate Kinase from Suifolobus acidcaldarius fused at the C-terminus with Sup35 Nterminal domain from Saccharomyces cerevisiae WO 2010/079357 PCT/GB2010/050018 43 SEQ ID 51 DNA sequence of Sup35N fused at the 5' end of Adenylate Kinase from Thermotoga maritime. SEQ ID 52 Protein sequence of Adenylate Kinase from Thermotoga maritima fused at the N-terminal with Sup35N. SEQ ID 53 DNA sequence of Sup35N fused at the 3' end of Adenylate Kinase from Thermotoga maritima. SEQ ID 54 Protein sequence of Adenylate Kinase from Thermotoga rintirna fused at the C-terminal with Sup35N SEQ ID 55 DNA sequence encoding a short Sup35 peptide capable of aggregating to form amyloid fibrils; for use as a fusion peptide with tAK genes. SEQ ID 56 Sup35 derived amyloid peptide SEQ ID 57 DNA sequence encoding a Norovirus capsid protein (58kDa) SEQ ID 58 Protein sequence of Norovirus capsid protein (58kDa) SEQ ID 59 DNA sequence for a synthetic gene encoding a Norovirus capsid protein (58kDa) optimised for expression in E.coli SEQ ID 60 DNA sequence for a synthetic gene encoding a Norovirus capsid protein (58kDa) optimised for expression in E.coli fused at the 5' end of a gene encoding the tAK from Thermotoga maritime. SEQ ID 61 Protein sequence of a Norovirus capsid protein (58kDa) fused at the N-terminus of the Adenylate Kinase from Thermotoga maritima, SEQ ID 62 Protein sequence of a bacteriophage MS2 coat protein SEQ ID 63 Protein sequence of a bacteriophage PP7 coat protein monomer SEQ ID 64 Protein sequence of a bacteriophage PP7 coat protein dimer SEQ ID 65 Protein sequence of Ecoli CsgA SEQ ID 66 Protein sequence of Salmonella AgfA SEQ ID 67 Protein sequence of adenylate kinase from ThermotogEa maritima fused to the N terminus of Ecoli CsgA SEQ ID 68 Protein sequence of the hydrophobin 3 protein from Fusarium species SEQ ID 69 Protein sequence of the hydrophobin 5 protein from Fusarium species WO 2010/079357 PCT/GB2010/050018 44 SEQ ID 70 Protein sequence of cement-like protein from Balanus albicostatus (19K) SEQ ID 71 Protein sequence of cement-Hike protein from Megabalanus rosa (20k) SEQ ID 72 Protein sequence of fusion of the barnacle protein from Balanus albicostatus with the tAK from Thermotoga maritma; N-terminal fusion SEQ ID 73 Protein sequence of fusion of the barnacle protein from Balanus atbicostatus with the tAK from Thermotogca maritime; C-terminal fusion SEQ ID 74 Protein sequence of Balanus albicostatus calcite-specific adsorbent SEQ ID 75 Protein sequence of a peptide derived from a barnacle cement protein SEQ ID 76 Protein sequence of a peptide derived from a barnacle cement protein SEQ ID 77 Protein sequence of a peptide derived from a barnacle cement protein SEQ ID 78 Protein sequence of adenylate kinase from Ecoli SEQ ID 79 Protein sequence of pyruvate kinase from E.coli SEQ ID 80 Protein sequence of acetate kinase from E.coli SEQ ID 81 Protein sequence of adenylate kinase from Methanococcus voltae (MVO) SEQ ID 82 Protein sequence of adenylate kinase from Methanococcus thermolithotrophicus (MTH). SEQ ID 83 Protein sequence of adenylate kinase from Bacillus globisporus SEQ ID 84 Protein sequence of adenylate kinase from Bacius subtiis WO 2010/079357 PCT/GB2010/050018 45 SEQUENCE LISTING SEQ ID NO:1 Met Lys lie Gly Ile Val Thr Gly lie Pro Gly Val Gly Lys Thr Thr Val Leu Ser Phe Ala Asp Lys Ile Leu Thr Gu Lys Gly lie Ser Lys lie Val Asn Tyr Gly Asp Tyr Met Leu Asn Thr Ala Leu Lys Gluy Tyr Val Lys Ser Arg Asp Gu lie Arg Lys Leu Gin lie Giu Lys Gin Arg Gu Leu Gin Ala Leu AL AL Arg Arg lie Val Glu Asp Leu Ser Leu Leu Gly Asp Giu Gly lie Giv Leu lie Asp Thr His Ala lie Arg Thr Pro Ala Gly Tyr Leu Pro Gly Leu Pro Arg His Val lie Glu Val Leu Ser Pro Lys Val lie Phe Leu Leu Glu Ala Asp Pro Lys lie lie Leu Gu Arg Gin Lys Arg Asp Ser Ser Arg Ala Arg Thr Asp Tyr Ser Asp Thr Ala Val lIe Asn Giu Val lie Gin Phe Ala Arg Tyr Ser Ala Met Ala Ser AL Val Leu Val Gly Ala Ser Val Lys Val Val Val Asn Gin Glu Gly Asp Pro Ser lie Ala Ala Ser Gu lie lie Asn Ser Leu Met SEQ ID NO: 2 Met Lys le Gly Ile Val Thr Gly lie Pro Gly Val Gly Lys Ser Thr Val Leu Ala Lys Val Lys GIu lie Leu Asp Asn Gin Glv le Asn Asn LVs lie Ile Asn Tyr Gly Asp Phe Met Leu Ala Thr Ala Leu Lys Leu Gly Tyr Ala Lys Asp Arg Asp Giu Met Arg Lys Leu Ser Val Gu Lys Gin LVs Lys Leu Gin lie Asp Ala Ala Lys Giy lie Ala Giu Glu Ala Arg Ala Gly Gly Giu Gly Tyr Leu Phe lIe Asp Thr His Ala Val lie Arg Thr Pro Ser Gly Tyr Leu Pro Gly Leu Pro Ser Tyr Val lIe Thr Glu Ile Asn Pro Ser Val lie Phe Leu Leu Glu Ala Asp Pro Lys lie lie Leu Ser Arg Gin Lys Arg Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp Glu Ser Val lie Leu Giu Thr lie Asn Phe Ala Arg Tyr AL AL Thr Ala Ser Ala Val Leu AL Gly Ser Thr Val Lys Val lie Val Asn Val Giu Gk Asp Pro Ser lie Ala Ala Asn Glu Ile lie Arg Ser Met Lys SEQ ID NO: 3 Met Ser Lys Met Lys lie Giv lie Val Thr GIy lie Pro Giv Val Gv Lys Thr Thr Val Leu Ser Lys Val Lys Glu Ile Leu Giu Glu Lys Lys lie Asn Asn Lys lie Val Asn Tyr Gly Asp Tyr Met Leu Met Thr Ala Met Lys Leu Gly Tyr Val Asn Asn Arg Asp Glu Met Arg Lys Leu Pro Val Glu Lys GIn Lys Gin Leu Gin lie Gu Ala Ala Arg Gy Ile Ala Asn Giu Ala Lys Glu Giy Gly Asp Gly Leu Leu Phe lie Asp Thr His Ala Val lie Arg Thr Pro Ser Gly Tyr Leu Pro Gly Leu Pro Lys Tyr Val lie Gu Glu lie Asn Pro Arg Val lie Phe Leu Leu Glu Ala Asp Pro Lys Val lie Leu Asp Arg GIn Lys Arg Asp Thr Ser Arg Ser Arg Ser Asp Tyr Ser Asp Giu1 Arg lie le Ser Glu Thr lie Asn Phe Ala Arg Tyr Ala Ala Met Ala Ser Ala Val Leu Val GIv Ala Thr Val Lys lie Val Ile Asn Val Glu Gly Asp Pro Ala /al Ala Ala Asn Glu lIe lie Asn Ser Met Leu SEQ ID NO: 4 Met Pro Phe Val Val lie lie Thr Gly lie Pro Gly Val Gly Lys Ser Thr lie Thr Arg Leu Ala Leu Gin Arg Thr Lys Ala Lys Phe Arg Leu lIe Asn Phe Gly Asp Leu Met Phe GILu Glu Ala Val Lys Ala Gly Leu Val Lys His Arg Asp Glu Met Arg Lys Leu Pro Leu Lys lIe Gn Arg Glu Leu Gin Met Lys Ala Ala Lys Lys le Thr Glu Met Ala Lys GIu His Pro lIe Leu Val Asp Thr His Ala Thr Ile Lys Thr Pro His Gly Tyr Met Leu Gly Leu Pro Tyr Giu Val Val Lvs Thr Leu Asn Pro Asn Phe lie Val lIe lie Glu Ala Thr Pro Ser Glu lie Leu Gly Arg Arg Leu Arg WO 2010/079357 PCT/GB2010/050018 46 Asp Leu Lys Arg Asp Arg Asp Val GIU Thr GIU Glu Gin lie Gin Arg His Gin Asp Leu Asrn Arg Ala Ala Ala lie Ala Tyr Ala Met His Ser Asn Ala Leu lie Lys Ile lIe Glu Asn His Gu Asp Lys Gly Leu Glu Gu Ala Val Asn Glu Leu Val Lys lie Leu Asp Leu Ala Val Asn Glu Tyr Ala SEQ ID NO: 5 Met Pro Phe Val Val Ile lie Thr Gly lie Pro GIy Val Gly Lys Ser Thr lie Thr Lys Leu Ala Leu Gin Arg Thr Arg AL Lys Phe Lys Leu lie Asn Phe Glv Asp Leu Met Phe Glu Giu Ala Leu Lys Leu Lys Leu Val Lys His Arg Asp Giu Met Arg Lys Leu Pro Leu Glu Val GIn Arg Giu Leu GIn Met Asn Ala Ala Lys Lys lie Ala Gu Met Ala Lys Asn Tyr Pro lie Leu Leu Asp Thr His AL Thr lIe Lys Thr Pro His Gly Tyr Leu Leu Gly Leu Pro Tyr Glu Val lie Lys lie Leu Asn Pro Asn Phe lie Val ie le Gu Ala Thr Pro Ser Glu Ile Leu Glv Arg Arg Leu Arg Asp Leu Lys Arg Asp Arg Asp Val Glu Thr GIu Glu Gin Ile Gin Arg His Ginr Asp Leu Asn Arg Ala Ala Ala liE Thr Tyr Ala Met His Ser Asn Ala Leu lie Lys lie lie Glu Asn His G3u Asp Lys Gly Leu Glu Glu Ala Val Asn Giu Leu Val Lys Ile Leu Asp Leu Ala Val Lys Gu Tyr Ala SEQ ID NO: 6 Met 3cr Phe Val Val lie lIe Thr Gly lIe Pro Gly Val Gly Lys Ser Thr lie Thr Arq Leu Ala Leu Gin Arg Thr Lys Ala Lys Phe Lys Leu lie Asn Phe Gly Asp Leu Met Phe Glu Giu Ala Val Lys Ala Gly Leu Val Asn His Arg Asp Glu Met Arg Lys Leu Pro Leu Giu lie Gin Arg Asp Leu Gin Met Lys Val Ala Lys Lys lie Ser Glu Met Ala Arg GIn Gin Pro le Leu Leu Asp Thr His Ala Thr lie LVs Thr Pro His Gly Tyr Leu Leu Gly Leu Pro Tyr Glu Val lie Lys Thr Leu Asn Pro Asn Phe lie Val lie lie Glu Ala Thr Pro Ser Glu lie Leu Gly Ar Arg Leu Arg Asp Leu Lys Arg Asp Arg Asp Val Gu Thr Glu Gliu Gin lIe Gin Arg His Gin Asp Leu Asn Arm AL AL Ala lie Ala TVr Ala Met His Ser Asn Ala Leu lie Lys lIe lie Giu Asn His Glu Asp Lys Gly Leu Glu Glu Ala Val Asn Glu Leu Val Glu Ile Leu Asp Leu Ala Val Lys Glu Tyr Ala SEQ ID NO: 7 Met Lys Asn Lys Leu Val Val Val Thr Gly Val Pro Gly Val Gly Gly Thr Thr lie Thr Gin Lys Ala Met Glu Lys Leu Ser Glu Glu Gly lie Asn Tyr Lys Met Val Asn Phe Glv Thr Val Met Phe Gu Val Ala Gin Glu Glu Asn Leu Val Glu Asp Arg Asp Girt Met Arg Lys Leu Asp Pro Asp Thr Gin Lys Arg lie Gin Lys Leu Ala Gly Arg Lys lie Ala Giu Met Val Lys Glu Ser Pro Val Val Val Asp Thr His Ser Thr lie Lys Thr Pro Lvs Gly Tyr Leu Pro Glv Leu Pro Val Trp Val Leu Asn Gli Leu Asn Pro Asp lie lie le Val Val Giu Thr Ser Gly Asp Glu lie Leu Ile Arg Arg Leu Asn Asp Giu Thr Arg Asn Arg Asp Leu Glu Thr Thr Ala Gly lie Glu Giu His Gin Ile Met Asn Arg Ala Ala Ala Met Thr Tyr Gly Val Leu Thr Gly Ala Thr Val Lys lIe lIe Gin Asn Lys Asn Asn Leu Leu Asp Tyr Ala Val Glu Gu Leu lie Ser Val Leu Arg SEQ ID NO: 8 Met Lys Asn Lys Val Val Val Val Thr Gly Val Pro Gly Val Gly Ser Thr Thr Ser Ser Gin Leu Ala Met Asp Asn Leu Arg Lys Giu Gly Val Asn Tyr Lys Met Val Ser Phe Gly Ser Val Met Phe Giu Val Ala Lys Giu Giu WO 2010/079357 PCT/GB2010/050018 47 Asn Leu Val Ser Asp Arg Asp Gin Met Arg Lys Met Asp Pro Glu Thr Gin Lys Arg lie Girt Lys Met Ala Gly Arg Lys lie Ala hGu Met Ala Lys Glu Ser Pro Val Ala Val Asp Thr His Ser Thr Val Ser Thr Pro Lys Gly Tyr Leu Pro Giy Leu Pro Ser Trp Val Leu Asr Gu Leu Asn Pro Asp Leu lie Ile Val Val GIU Thr Thr Gly Asp Glu lie Leu Met Arg Arg Met Ser Asp Glu Thr Arg Val Arg Asp Leu Asp Thr Ala Ser Thr lie Glu Gn His GIn Phe Met Asn Arg Cys Ala Ala Met Ser Tyr Gly Val Leu Thr Gly Ala Thr Val Lys lHe Val Gin Asn Arg Asn Gly Leu Leu Asp GIn Ala Val Gu Glu Leu Thr Asn Val Leu Arg SEQ ID NO: 9 Met Met Met Met Lys Asn Lys Val Val Val lie Val Giy Val Pro Gly Val Gly Ser Thr Thr Val Thr Asn Lys Ala lie Glu Glu Leu Lys Lys Gu Gly le Glu Tyr Lys lie Val Asn Phe Gv Thr Val Met Phe Gu lie Ala Lys Glu Glu Gly Leu Val Giu His Arg Asp Gin Leu Arg Lys Leu Pro Pro GILu Gu Gin Lys Arg lie Gin Lys Leu Ala G ly Lys Lys lie Ala Gu Met Ala Lys Glu Phe Asn lie Val Val Asp Thr His Ser Thr lie Lys Thr Pro Lys Gly Tyr Leu Pro Gly Leu Pro Ala Trp Val Leu Gu Giu Leu Asn Pro Asp lie lie Val Leu Val Giu Ala Giu Asn Asp Glu Ile Leu Met Arq Arg Leu Lys Asp Gu Thr Arg Gin Arg Asp Phe Glu Ser Thr Giu Asp lie Gly Gu His lIe Phe Met Asn Arg Cys Ala Ala Met Thr Tyr Ala Val Leu Thr Gly Ala Thr Val Lys lIe lIe Lys Asn Arg Asp Phe Leu Leu Asp Lys Ala Val Gin Giu Leu Ile Giu Val Leu Lys SEQ ID NO: 10 Met Giv Tyr Val lie Val Ala Thr Gly Val Pro Gly Val Gly Ala Thr Thr Val Thr Thr Gu Ala Val Lys Glu Leu Glu Gly Tyr Gu His Val Asn Tyr Gly Asp Val Met Leu Gu lIe Ala Lys Giu Gu Giv Leu /al Glu His Arg Asp Glu lIe Arg Lys Leu Pro Ala Giu Lys Gin Arg Glu lIe Gin Arg Leu Ala Ala Arg Arg lie Ala Lys Met Ala Giu Gu Lys Gu Gly lie lie Val Asp Thr His Cys Thr lIe Lys Thr Pro Ala Gly Tyr Leu Pro Gly Leu Pro lie Trp Val Leu Giu Giu Leu Gin Pro Asp Val Ile Val Leu lIe Giu Ala Asp Pro Asp Gu lIe Met Met Arg Arg Val Lys Asp Ser Giu Giu Arq Gin Arg Asp Tvr Asp Arg Ala His GIu lie Glu Giu His Gin Lys Met Asn Arg Met Ala Ala Met Ala Tyr AL Ala Leu Thr Gly Ala Thr Val Lys lie lie Giu Asn His Asp Asp Arg Leu Giu Glu Ala Val Arg Giu Phe Val Glu Thr Val Arg Ser Leu SEQ ID NO: 11 Met Lys Asn Lys Val Val Val Val Thr GLy Val Pro Giy Val GV Gly Thr Thr Leu Thr Gin Lvs Thr lIe Gu Lys Leu Lys Gu Gliu Glv lie Gu Tyr Lys Met Val Asn Phe Glv Thr Val Met Phe Giu Val Ala Lys Giu Giu Gly Leu Val Glu Asp Arg Asp GIn Met Arg Lys Leu Asp Pro Asp Thr Gin Lys Arg Ile GIn Lys Leu Ala Gl, Arg Lys lie Ala GIu Met Ala Lys GIu Ser Asn Val Ile Val Asp Thr His Ser Thr Val Lys Thr Pro Lys Gly Tyr Leu Ala Gly Leu Prc lie Trp Val Leu Glu Glu Leu Asn Pro Asp lie lie Val Ile Val Gilu Thr Ser Ser Asp Glu Ile Leu Met Arg Arg Leu Gly Asp Ala Thr Arg Asn Arg Asp lIe Giu Leu Thr Ser Asp lIe Asp Gi His Gin Phe Met Asn Arg Cys Ala Ala Met Ala Tyr Gv Val Leu Thr GV Ala Thr Val Lys lie Ile Lys Asn Arg Asp Gly Leu Leu Asp Lys Ala Val Giu Giu Leu lIe Ser Val Leu Lys WO 2010/079357 PCT/GB2010/050018 48 SEQ ID NO: 12 Met Lys lie Val Ile Val Ala Leu Pro Gly Ser Giy Lys Thr Thr lie Leu Asn Phe Val Lys Gin Lys Leu Pro Asp Val Lys lie Val Asn Tyr GIy Asp Val Met Leu Giu lie Ala Lys Lys Arg Phe Gly lie Gin His Arg Asp Gu Met Arg Lys Lys lIe Pro Val Asp Giu Tyr Arg Lys Val Gin Giu Glu Ala Ala Giu Tyr lie Ala Ser Leu Thr Gly Asp Val Ile lie Asp Thr His Ala Ser lie Lys lie Gly Gly Gly Tyr Tyr Pro Gly Leu Pro Asp Arg Ile Ile Ser Lys Leu Lys Pro Asp Val lie Leu Leu Leu Glu Tyr Asp Pro Lys Val lie Leu Glu Arg Arg Lys Lys Asp Pro Asp Arg Phe Arg Asp Leu Giu Ser Gu Glu Giu lie Glu Met His Gin GIn Ala Asn Arg Tyr Tyr Ala Phe Ala Ala Ala Asn Ala GIv Glu Ser Thr Val His Val Leu Asn Phe Arg Gly Lys Pro Glu Ser Arg Pro Phe Glu His Ala Giu Val Ala Ala Glu Tyr lie Val Asn Leu Ile Leu Arg Thr Arg GIn Lys Ser SEQ ID NO: 13 Met Met Ala Tyr Leu Val Phe Leu GIy Pro Pro GIy Ala Gly Lys Gly Thr 1 yr Ala Lys Arg lie GIn Gku Lys Thr Gly Ile Pro His lie Ser Thr Gly Asp lie Phe Arg Asp lIe Val Lys Lys Glu Asn Asp Giu Leu Gly Lys Lys lie Lys Glu lie Met Giu Lys Gly Gu Leu Val Pro Asp Gu Leu Val Asn Glu Val Val Lys Arg Arg Leu Ser Giu Lys Asp Cys Giu Lys Gly Phe lie Leu Asp Gly Tyr Pro Arg Thr Val Ala Gin Ala Giu Phe Leu Asp Ser Phe Leu Glu Ser Gin Asn Lys Gn Leu Thr AL Ala Val Leu Phe Asp Val Pro Gu Asp Val Val Val GIn Arg Leu Thr Ser Arg Arg lie Cys Pro Lys Cys Glv Arg lie Tyr Asn Met lie Ser Leu Pro Pro Lys Glu Asp Giu Leu Cys Asp Asp Cys Lys Val Lys Leu Val Gin Arg Asp Asp Asp Lys Gu Glu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Gu Lvs Thr Gin Pro Val lie Asp Tyr Tyr GlV Lys Lys (Thy lie Leu Lys Arg Val Asp Gly Thr lie Gly lie Asp Asn Val Val Ala Gu Val Leu Lys lie lie Gly T rp Ser Asp Lys SEQ ID NO: 14 Met Lys Val Arg His Pro Phe Lys Val Val Val Val Thr Gly Val Pro Gly Val Gly Lys Thr Thr Val lie Lys Giu Leu GIn Gly Leu Ala Giu Lys Gu Gly Val Lys Leu His lie Val Asn Phe Gly Ser Phe Met Leu Asp Thr AL Val Lys Leu Gly Leu VI Giu Asp Arg Asp Lys lie Arg Thr Leu Pro Leu Arg Arg GIn Leu Giu Leu Gin Arg Giu Ala Ala Lys Arg Ile Val Ala Gu Ala Ser Lys AL Leu Gly Gly Asp Gly Val Leu lie lie Asp Thr His Ala Leu Val Lys Thr Val Ala Gly Tyr Trp Pro Gly Leu Pro Lys His Val Leu Asp Giu Leu Lys Pro Asp Met Ile Ala Val Val Glu Ala Ser Pro Giu Glu Vd Ala Ala Arg Gin Ala Arg Asp Thr Thr Arg T yr Arg Val Asp le Gly Gly Val GIU Gly Val Lys Arg Leu Met Glu Asn Ala Arg Ala Ala Ser Ile Ala Ser Ala ie GIn Tyr Ala Ser Thr Val Ala lie Val Giu Asn Arg Glu Gly Glu Ala Ala Lys Ala Ala Glu Glu Leu Leu Arg Leu lie Lys Asn Leu SEQ ID NO: 15 Met Asn Leu le Phe Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Gin Ala Lys Arg Val Ser Glu Lys Tvr GlV lie Pro Gin lie Ser Thr Gy Asp Met Leu Arg Glu Ala Val Ala Lys Gy Thr Glu Leu Gly Lys Lys Ala Lys Glu Tyr Met Asp Lys Gly GILu Leu Val Pro Asp GIU Val Val le Gly Ile Val Lys Glu Arg Leu Gin GIn Pro Asp Cys Glu Lys Gly Phe lie Leu Asp Gy Phe Pro Arg Thr Leu Ala Gin Ala Glu Ala Leu Asp Glu Met Leu Lys Gu Leu Asn Lys Lys lIe Asp Ala Val Ile Asn Val Val Val Pro Giu Giu Glu Val Val Lys Arg ie Thr Tyr Arg Arg Thr WO 2010/079357 PCT/GB2010/050018 49 Cys Arg Asn Cys Gly Ala Val Tyr His Leu lie Tyr Ala Pro Pro Lys Glu Asp Asr Lys Cys Asp Lys Cys Gly Giy Glu Leu Tyr Gin Arg Asp Asp Lys Giu Gu Thr Val Arg Glu Arg Tyr Arg Val Tyr Lys Gin Asn Thr GIu Pro Leu lie Asp Tyr Tyr Arg Lys Lys Gly lie Leu Tyr Asp Val Asp Gly Thr Lys Asp lie Gi Gly Val Trp Lys Gu lie Glu Ala lie Leu Glu Lvs lie Lys Ser SEQ ID NO: 16 Met Asn lie Leu le Phe Gly Pro Pro Ghy Ser Gly Lys Ser Thr Gin Ala Arg Arg lie Thr Gki Arg Tyr Gly Leu Thr Tyr lie Ala Ser Gly Asp lie lie Arg Ala Giu Ile Lys Ala Arg Thr Pro Leu Gly lie Giu Met Giu Arq Tyr Leu Ser Arg Gly Asp Leu lie Pro Asp Thr Ile Val Asn Thr Leu le L Ser Lys Leu Arg Arg Val Arg Giu Asn Phe lie Met Asp Gly Tyr Pro Arg Thr Pro Giu Gin Val Ile Thr Leu Giu Asn Tyr Leu Tyr Asp His Gly lie Lys Leu Asp Val Ala lie Asp lie Tyr Hle Thr Lys GIU Glu Ser Val Arg Arg Ile Ser Gly Arg Arg lie Cys Ser Lys Cys Gly Ala Val Tyr His Val Gu Phe Asn Pro Pro Lys Val Pro Gly Lys Cys Asp Ile Cys Gly Gly Gu Leu lie GTIn Arg Pro Asp Asp Arg Pro Gki lie Val Giu Lys Arg Tyr Asp lie Tyr Ser Lys Asn Met Gu Pro lie lie Lys Phe Tyr Gin Lys Gin Gly lie Tyr /al Arg lie Asp Giy His Ghy Ser lie Asp Gu Val Trp Giu Arg Ile Arg Pro Leu Leu Asp Tyr Ile Tyr Asn Gin Gki Asn Arg Arg SEQ ID NO: 17 Met Pro Phe Val Val le lie Thr Gly lie Pro Gly Val Gly Lys Ser Thr lie Thr Arg Leu Ala Leu Gin Arg Thr Lys Ala Lys Phe Arg Leu lie Asn Phe Gly Asp Leu Met Phe Giu Gki Ala Val Lys AL( Gly Leu Val Lys His Arg Asp Glu Met Arg Lys Leu Pro Leu Xaa lie Gin Arg Giu Leu Gin Met Lys Ala Ala Lys Lys Ile Xaa Giu Met AL Lys Gliu His Pro lie Leu Val Asp Thr His Ala Thr lie Lys Thr Pro His Gly Tyr Xaa Leu Gly Leu Pro Tyr Gu /al /al Lys Thr Leu Asn Pro Asmn Phe ile /al Ile Ile Giu Ala Thr Pro Ser Gu lie Leu Gly Arg Arg Leu Arg Asp Leu Lys Arg Asp Arg Asp Val Giu Thr Gu Glu Gin Ile Gin Arg His Gin Asp Leu Asn Arg Ala Ala Ala lie Xaa Tvr Ala Met His Ser Asn Ala Leu lie LVs lie lie Gu Asn His Glu Asp Lys Giy Leu Gu Gu Ala Val Asn Glu Leu Val Lys ie Leu Asp Leu Ala Val Asn GIu Tyr Ala SEQ ID NO: 18 Met Pro Phe Val Val lie lie Thr Gly lie Pro Gly Val Gly Lys Ser Thr lie Thr Lys Leu Ala Leu Gin Arg Thr Arg Ala Lys Phe Lys Leu Ile Asn Phe Gly Asp Leu Met Phe GIu Giu Ala Leu Lys Leu Xaa Leu Val Lys His Arg Asp Gu Met Arg Lys Leu Pro Leu Giu Val Gin Arg Giu Leu Gin Met Asn Ala Ala Lys Lys lie Ala Giu Met AL Lys Asn Tyr Pro lie Leu Leu Asp Thr His Ala Thr lie Lys Thr Pro His Gy Tyr Leu Leu Gly Leu Pro Tyr GIu Val lie Lys lie Leu Asn Pro Asn Phe lie Val Ile lie Glu Ala Thr Pro Ser Giu lie Leu Gly Arg Arg Leu Arg Asp Leu Lys Arg Asp Arm Asp Val Gu Thr Gu Glu Gin lie GIn Arg His Gin Asp Leu Asn Arg Ala Ala Ala Ile Xaa Tyr Ala Met His Ser Asr Ala Leu Ile Lys Ile Ile Glu Asn His GIU Asp Lys Gly Leu Glu Giu Ala Val Asn Gu Leu Val Lys lie Leu Asp Leu Ala Val Lys Glu Tyr Ala SEQ ID NO: 19 WO 2010/079357 PCT/GB2010/050018 50 Met Lys Hle Glv lie Val Thr Glv le Pro Gly Val Gly Lys Ser Thr Val Leu Ala Lys Val Lys Glu Ile Leu Asp Asn Gin Gly Ile Asn Asn Lys lie lie Asn Tyr Gly Asp Phe Met Leu Ala Thr Ala Leu Lys Leu Gly Tyr Ala Lys Asp Arg Asp Giu Met Arg Lys Leu Ser Val Gu Lys Grt Lys Lys Leu Girt lie Asp Ala Ala Lys Gly Ile Ala Giu Gu Ala Arg Ala Gkiy Gly Gu Gly Tyr Leu Phe Ile Asp Thr His Ala Val lie Arg Thr Pro Ser Gly Tyr Xaa Pro Gly Leu Pro Ser Tyr Val lIe Thr Giu lIe Asn Pro Ser Val le Phe Leu Leu Giu Ala Asp Pro Lys lIe lie Leu Ser Arg Gin Lys Arg Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp Glu Ser Val Ile Leu Glu Thr lIe Asn Phe Ala Arg Tyr Ala Ala Thr AL Ser Ala Val Leu Ala Gly Ser Thr Val Lys Val lie Val Asn Val Giu Gly Asp Pro Ser lie Ala Ala Asn Glu lie lie Arg Ser Met Lys SEQ ID NO: 20 Met Arg Val Leu Val lie Asn Ser Gly Ser Ser Ser lIe Lys Tyr Gin Leu lIe Giu Met Giu Gly Giu Lys Val Leu Cys Lys Gly Ile Ala GIu Arg Ile Gly Ile Giu Gly Ser Arg Leu Val His Arg Val Glv Asp Giu Lvs His Val le Gu Arg 31u Leu Pro Asp His Gu G1u Ala Leu Lys Leu lie Leu Asn Thr Leu Val Asp Gu Lys Leu Gly Val lie Lys Asp Leu Lys Glu lie Asp Ala /al Gly His Arg /al aI His Gly GlV Glu Arg Phe Lys Giu Ser /al Leu Val Asp Glu Giu Val Leu Lys Ala lIe Gu Giu Val Ser Pro Leu Ala Pro Leu His Asn Pro AL Asn Leu Met Gly Ile L ys Ala Ala Met Lys Leu Leu Pro Gly Val Pro Asn Val Ala /aI Phe Asp Thr Ala Phe His Gin Thr lIe Pro GIn Lys Ala Tyr Leu Tyr Ala lie Pro Tyr Gu Tyr Tyr Glu Lys Tyr Lys Ie Arg Arg Tyr Gy Phe His Giy Thr Ser His Arg Tyr Val Ser Lys Arg Ala Ala Glu Ile Leu Gly Lys Lys Leu Gu Glu Leu Lys lIe lie Thr Cys His lie GlV Asn Giv Ala Ser Val Ala Ala Val Lys Tyr Gly Lys Cys Val Asp Thr Ser Met Gly Phe Thr Pro Leu Giu Gly Leu Val Met Gly Thr Arg Ser Gly Asp Leu Asp Pro Ala Ile Pro Phe Phe lIe Met Giu Lys Giu Gly Ile Ser Pro Gin Gu Met Tyr Asp lie Leu Asn Lys Lys Ser Gly Val Tyr Gly Leu Ser Lys Giy Phe Ser Ser Asp Met Arg Asp lie Gliu Gu Ala Ala Leu LVs Gly Asp Glu Trp Cys Lvs Leu Val Leu Giu lie Tyr Asp Tyr Arg lIe Ala Lys Tyr Ile Gly Ala Tyr Ala Ala Ala Met Asn Gly Val Asp Ala lIe Val Phe Thr Ala Gy Val Glv Glu Asn Ser Pro lie Thr Arg Giu Asp Val Cys Ser Tyr Leu Giu Phe Leu Gly Val Lys Leu Asp Lys Gin Lys Asn Glu Giu Thr lie Arg Gly Lys Glu Gly Ile lie Ser Thr Pro Asp Ser Arg Val Lys Val Leu Val Val Pro Thr Asn Glu Giu Leu Met lIe Ala Arg Asp Thr Lys Glu lIe /al Giu Lys lIe Gly Arg SEQ ID NO: 21 Met Arg Arg Met Lys Leu Prc Ser His Lys Thr Lys lie Val Ala Thr lIe Gly Pro Ala Thr Asn Ser Lys Lys Met lle Lys Lys Leu le Giu Ala Gly Met Asn Val Ala Arg Ile Asn Phe Ser His Gly Thr Phe Giu Giu His Ala Lys lie lIe Gu Met Val Arg Giu Gin Ser Gin Lys Leu Asp Arg Arg Val Ala lIe Leu Ala Asp Leu Pro Gly Leu Lys lIe Arm Val Gly Giu lIe Lys Gly Gly Tyr Val Glu Leu Giu Arg Gly Giu Lys Val Thr Leu Thr Thr Lys Asp lie Giu Gy Asp Giu Thr Thr lie Pro Val Gu Tyr Lys Asp Phe Pro Lys Leu /al Ser Lys Gly Asp /al lie Tyr Leu Ser Asp Gly Tyr lie Val Leu Arg Val Giu Asp Val Lys Giu Asn GIu Val Glu Ala Val Val lie Ser Giv Gly Lys Leu Phe Ser Arg Lys Giy lie Asn le Pro Lys Aia Tyr Leu Pro Val Giu Ala Ile Thr Pro Arg Asp lie Glu Ile Met Lys Phe Ala lie Giu His Gly Val Asp Ala lie Gly Leu Ser Phe Val Gly Asn V r Asp Val Leu Lys Ala Lys Ser Phe Leu Gu Arg Asn Gly Ala Gly Asp Thr Phe Val lie Ala Lys lie Giu Arg Pro Asp Ala Val Arg Asn Phe Asn Giu lie Leu Asn Ala Ala Asp Gly lie Met Ile Ala Arg Gly Asp Leu Gly Val Glu Met Pro lie Giu Gin WO 2010/079357 PCT/GB2010/050018 51 Leu Pro lie Leu Gin Lys Arg Leu lie Arg Lys Ala Asn Met Giu Gly Lys Pro Val lIe Thr Ala Thr Gin Met Leu Val Ser Met Thr Met Giu Lys Val Pro Thr Arg Ala Glu Val Thr Asp Val Ala Asn Ala lie Leu Asp Gly Thr Asp ALa Val Met Leu Ser Giu GIU Thr Ala Val Gly Lys Phe Pro Ile GIU Ala Val Glu Met Met Ala Arg lie Ala Lys Val Thr Glu Giu Tyr Arg Gu Ser Phe Gly lie Thr Arg Met Arg Giu Phe Leu Glu Gly Thr Lys Arg Giy Thr Hle Lys Giu Ala lie Thr Arg Ser lie lie Asp Ala ile Cys Thr lIe Gly Ile Lys Phe lie Leu Thr Pro Thr Lys Thr Gly Arg Thr Ala Arg Leu lie Ser Arg Phe Lys Pro Lys Gin Trp lie Leu Ala Phe Ser Thr Arg Gu Lys Val Cys Asn Asn Leu Met Phe Ser Tyr Gly Val Tyr Pro Phe Cys Met Glu Glu Gly Phe Asn Glu Asn Asp lie Vai Arg Leu Ile Lys Gly Leu Gly Leu /al Gly Ser Asp Asp Ile /al Leu Met Thr Giu GIy Lys Pro lie Gu Lys Thr Val Giy Thr Asn Ser lie Lys lie Phe Gin Ile Ala SEQ ID NO: 22 Met Arg Lvs Thr Lys lie Val Ala Thr Leu Gly Pro Ser Ser Gu Gu Lys Val Lys Gu Leu Ala Gu Tyr Val Asp Vai Phe Arg lie Asn Phe Ala His Gl, Asp Giu Thr Ser His Arg Lys Tyr Phe Asp Leu lie Arg Thr Tyr Aka Pro Giu Ser Ser lIe le /al Asp Leu Pro Gly Pro Lys Leu Arg Leu Gly Glu Leu Lys Glu Pro lie Giu Val Lys Lys Gly Asp Lys lIe Val Phe Ser Gin Lys Asp Gly lie Pro Val Asp Asp Glu Leu Phe Tyr Ser Ala Val Lys Giu Asn Ser Asp lie Leu lie Ala Asp Gly Thr lie Arg Val Arq Val Lys Ser Lys Ala Lys Asp Arg Val Gki Gly Thr Vai Ile Glu Giy Giv Ile Leu Leu Ser Arg Lys GIy lie Asn lie Pro Asn Val Asn Leu Lys Ser GIy lie Thr Asp Asn Asp Leu Lys I. eu Leu Lys Arg Ala Leu Asp Leu Gly Ala Asp Tyr lie GIy Leu Ser Phe Val lie Ser Giu Asn Asp Vai Lys Lys Val Lys Giu Phe Vai Gly Asp Glu Aka Trp Val Ile Ala Lys lie Glu Lys Ser Giu AL Leu Lys Asn Leu Thr Asn ile Val Asn Glu Ser Asp Gly lie Met Val Ala Arg GIv Asp Leu Ghy Val Giu Thr Gly Leu Gu Asn Leu Pro Leu lie GIn Arg Arg le Val Arg Thr Ser Arg Val Phe Gly Lys Pro Val lie Leu Ala Thr Gn Val Leu Thr Ser Met lie Asn Ser Pro lie Pro Thr Arg Ala Giu lie Ile Asp lIe Ser Asn Ser lie Met Gin Gly Val Asp Ser le Met Leu Ser Asp Glu Thr Ala lie( Gly Asn T yr Pro Val Giu Ser Val Arg Thr Leu His Asn le Ile Ser Asn /al Giu Lys Ser Val Lvs His Arg Pro ile Gly Pro Leu Asn Ser Giu Ser Asp Ala lIe Ala Leu Ala Ala Val Asn Ala Ser Lys Val Ser Lys Ala Asp Val lie Val Val Tyr Ser Arg Ser Gly Asn Ser lIe Leu Arg Val Ser Arg Leu Arg Pro Gu Arq Asn Ile lie Gy Val Ser Pro Asp Pro Arg Leu Ala Lys Lys Phe Lys Leu Cys Tyr Gly Val lie Pro lie Ser lie Asn Lys Lys Met Gin Ser lIe Asp Giu Ile Ile Asp Val Ser Ala Lys Leu Met Gin Giu Lys lie Lys Asp Leu Lys Phe Lys Lys lIe Val lie Val Gly Gly Asp Pro Lys Gin GIU Ala Gly Lys Thr Asn Phe Val lie Val Lys Thr Leu Glu Gin Gin Lys Lys SEQ ID NO: 23 Met Arq Ser Thr Lys lie Val Cys Thr Val Gly Pro Arg Thr Asp Ser Tyr Cii Met lIe Glu Lys Met Ie Asp Leu Gly Val Asn Val Phe Arg le Asn Thr Ser His Gly Asp Trp Asn Glu Gin Giu Gin Lys lie Leu Lys Ile Lys Asp Leu Arg Giu Lys Lys Lys Lys Pro Val Ala Ile Leu lie Asp Leu Ala Gly Pro Lys lie Arg Thr Gly Tyr Leu Giu Lys Glu Phe Val Glu Leu Lys Glu Gly Gin lie Phe Thr Leu Thr Thr Lys Glu lie Leu Gly Asn Glu His Ile Val Ser Val Asn Leu Ser Ser Leu Pro Lys Asp Val Lys Lys Gly Asp Thr lie Leu Leu Ser Asp Gly Giu lie Val Leu Gu Val Ie Glu Thr Thr Asp Thr Gu Val Lys Thr Val Val Lys Val Gy Gly Lys lie Thr His Arg Arg Gly Val Asn Val Pro Thr Ala Asp Leu Ser Val Giu Ser lie Thr Asp Arg Asp Arg GIu Phe lie Lys Leu Gly Thr Leu WO 2010/079357 PCT/GB2010/050018 52 His Asp Val GIu Phe Phe Ala Leu Ser Phe Val Arg Lys Pro Giu Asp Val Leu Lys Ala Lys Glu Glu Ile Arg Lys His Gly Lys Gu Ile Pro Val Ile Ser Lys lIe Gu Thr Lys Lys Ala Leu Glu Arg Leu Glu Glu le le Lys Val Ser Asp Gly lie Met Val Ala Arq Ghy Asp Leu Gly Val GII Ile Pro lie Gu Giu Val Pro lie Val Gin Lys Glu lIe lIe Lys Leu Ser Lys Tyr Tyr Ser Lys Pro Val Ile Val Ala Thr Gin Ile Leu Glu Ser Met lIe Glu Asn Pro Phe Pro Thr Arg Ala Glu Val Thr Asp lie Ala Asn Ala lie Phe Asp Gly Ala Asp Ala Leu Leu Leu Thr Ala Giu Thr Ala Val Gly Lys His Pro Leu Glu Ala Ile Lys Val Leu Ser Lys Val Ala Lys Glu Ala Giu Lys Lys Leu Glu Phe Phe Arg Thr lie Giu Tyr Asp Thr Ser Asp lie Ser Giu Ala lie Ser His Ala Cys Trp Gin Leu Ser Giu Ser Leu Asn Ala Lys Leu lie lIe Thr Pro Thr lie Ser Gly Ser Thr Ala Val Arg Val Ser Lys Tyr Asn Val Ser Gin Pro lie Val Ala Leu Thr Pro Gu Glu Lys Thr Tyr Tyr Arg Leu Ser Leu Val Arg Lys Val lie Pro Val Leu Ala Gu Lys Cvs Ser Gin Giu Leu Glu Phe lie Gu Lys Gly Leu Lys Lys Val Glu GIu Met Glv Leu Ala Giu Lys Gly Asp Leu Val Val Leu Thr Ser Gly Val Pro Gly Lys Val Gly Thr Thr Asn Thr lie Arg Val Leu Lys Val Asp SEQ ID NO: 24 Met Arg Arg al Lys Leu Pro Ser His Lvs Thr Lys Ile Val Ala Thr lie Gly Pro Ala Thr Asn Ser Arg Lys Met lie Lys Gin Leu lie Lvs Ala GBy Met Asn Val Ala Arg le Asn Phe Ser His Gy Ser Phe Glu Gu His Ala Arg Val Ile Glu lie iie Arg Gu Giu Ala Gin Lys Leu Asp Arg Arg Val Ala lie Leu Ala Asp Leu Pro Gly Leu Lys lie Arg Val Gly Gu lIe Lys GlyK Gly Tyr Val Glu Leu Lys Arg Gly Glu Lys Val lie Leu Thr Thr Lys Asp Val Glu Gly Asp Giu Thr Thr lie Pro Val Asp Tyr Lys Gly Phe Pro Asn Leu Val Ser Lys Gly Asp lie lie Tyr Leu Asn Asp Gy Tyr lie Val Leu Lys Val Giu Asn Val Arg Giu Asn Gu Val Glu Ala Val Val Leu Ser GlV GV Lys Leu Phe Ser Arg Lys Gly Val Asn lie Pro Lys Ala Tyr Leu Pro Val Glu Ala lie Thr Pro Lys Asp Phe Glu Ile Met Lys Phe Ala Ile Glu His Gly Val Asp Ala lie GTy Leu Ser Phe Val Gly Ser Val Tyr Asp Val Leu Lys Ala Lys Ser Phe Leu Glu Lys Asn Asn Ala Glu Asp Val Phe /al le Ala Lys lie Giu Arg Pro Asp Ala Val Arg Asn Phe Asp Gu lIe Leu Asn Ala Ala Asp Gly lie Met le Ala Arg GIy Asp Leu Gly Val G3u Met Pro le G1u Gin Leu Pro lIe Leu Gin Lvs Lys Leu lie Arg Lys Ala Asn Met Giu Gly Lys Pro Val lIe Thr Ala Thr Gin Met Leu Val Ser Met Thr Thr Glu Lys Val Pro Thr Arg Ala Gu Val Thr Asp /al Ala Asn Ala le Leu Asp Gly Thr Asp Ala Val Met Leu Ser Gu Gliu Thr Ala lIe Gly Lys Phe Pro lie Giu Thr Val Giu Met Met Gly Lys lie Ala Lys Val Thr Gu Giu Tyr Arg Gu Ser Phe Gly Leu Ser Arg Ile Arg Giu Phe Met Glu Ile Lys Lys Gly Thr lie Lys GIu Ala lie Thr Arg Ser lie lie Asp Ala lie Cys Thr lie Asp lie Lys Phe lie Leu Thr Pro Thr Arg Thr GMy Arg Thr Ala Arg Leu lie Ser Arg Phe Lys Pro Lys Gin Trp lie Leu Ala Phe Ser Thr Asn Gu Arg Val Cys Asn Asn Leu Met Phe Ser Tyr Gly Val Tyr Pro Phe Cys Leu GIiu Glu Gly Phe Asp Glu Asn Asp lie Val Arg Leu lie Lvs Gy Leu Glv Leu Val Gu Ser Asp Asp Met Val Leu Met Thr Glu Gly Lys Pro lie Gu Lys Thr Val Giv Thr Asn Ser lie Lys lie Phe Gin lie Ala SEQ ID NO: 25 Met Lys Val Leu Val lie Asn Ala Gly Ser Ser Ser Leu Lys Tyr Gin Leu lie Asp Met Thr Asn Gu Ser Ala Leu Ala Val Gly Leu Cys GIu Arg Ile Gly lIe Asp Asn Ser lie lie Thr Gin Lys Lys Phe Asp Gly Lys Lys Leu Giu Lys Leu Thr Asp Leu Pro Thr His Lys Asp Ala Leu Glu Glu Val Val Lys Ala Leu Thr Asp Asp Gu Phe Gly Val lIe Lys Asp Met Gly Giu lie Asn Ala Val Gly His Arg Val Val His Gly Gly Giu Lys Phe Thr Thr Ser Ala WO 2010/079357 PCT/GB2010/050018 53 Leu Tyr Asp GIu Gly /al Gu Lys Ala lie Lys Asp Cys Phe Glu Leu Ala Pro Leu His Asn Pro Pro Asn Met Met Gly lie Ser Ala Cys Ala Gu lie Met Pro Gly Thr Pro Met Val lie Val Phe Asp Thr Ala Phe His Gin Thr Met Pro Pro Tyr Ala Tyr Met Tyr Ala Leu Pro Tyr Asp Leu Tyr Glu Lys His Gly Val Arg Lys Tyr Gly Phe His Giv Thr Ser His Lys Tyr Vl Ala Glu Arg Ala Ala Leu Met Leu GTy Lys Pro Ala Glu Gu Thr Lys lie lie Thr Cys His Leu Gly Asn Gly Ser Ser lIe Thr Ala al GIu Gly Gly Lys Ser Val Glu Thr Ser Met Gly PhE Thr Pro Leu Gu Gly Leu Ala Met Gly Thr Arg Cys Gly Ser lie Asp Pro Ala lie Val Pro Phe Leu Met Gu Lys Glu Gly Leu Thr Thr Arg Giu lie Asp Thr Leu Met Asn Lys Lys Ser Gly Val Leu Gly Val Ser Gly Leu Ser Asn Asp Phe Arg Asp Leu Asp Glu Ala Ala Ser Lys Gly Asn Arg Lys Ala Glu Leu Ala Leu Giu lie Phe Ala Tyr Lys /ai Lys Lys Phe lie Gly Giu Tyr Ser Ala Val Leu Asn Gly Ala Asp Ala Val Val Phe Thr Ala Glv lie Gly G(u Asn Ser Ala Ser lie Arg Lys Arm Ile Leu Thr Gly Leu Asp Gly lie Gly lie Lys lIe Asp Asp Glu Lys Asn Lys lie Arg Gy GIn Glu lie Asp lie Ser Thr Pro Asp Ala Lys /ai Arg Val Phe Val lie Pro Thr Asn Gu Gu Leu Ala lie Ala Arg Gu Thr Lys Glu lIe Val Gu Thr Glu /al Lys Leu Arg Ser Ser lie Pro /al SEQ ID NO: 26 atgaagattg gtattgtaac tggaattcct ggtgtaggga aaagtactgt cttggctaaa gtaaagaga tattggataa tcaaggta ta aatacaaga tcataaatta tggagatttt atgttagcaa cagcattaaa attagqtat gctaqaata gaqacgaaat gagaaaatta tctgtagaaa agcagaagaa attgcagatt gatgCggcta aaggtatag tgaagaggca agagcaggtg gagaaggata tctittcata gatacgcatg ctqtgatacg tacaccctct ggataittac ctggtftacc gtcatatgta attacagaaa taaatecgte tgttatcttt ttactgpaag ctgatcctaa gataatatta tcaaggc:aaa agagagatac aacaaggaat agaaatgatt atagtgacga atcagttata ttagaaacca taaaCtte tagatatgca gctactgctt Ctgcagtatt agccppttet actgttaagg taattgtaaa cgtggaagga giatectagta tpagtaa tgagataata aggtctatga agtaa SEQ ID NO: 27 atgaaaatcg qtatoqttac cggtatcccg ggtgtggta aatctaccgt tctggctaaa qttaaagaaa tcctggacaa ccagggtatc aacaacaaaa teatcaacta cggtgacttc atgCtggcta ccgctctaa actgggttac gctaaapacc gtgacgaat gcgtaaactg tctgttgaaa aacagaaaaa actgcagatc gacgetgcta apptatope tgaagaagct cptgctpptg gtgaagptta cotgttate gacacccac ctgttatccg tacccgtct ggttacctge cgggtetgcc gtcttacgtt atcaccgaaa tcaacccgtc tgttatcttc ctgctggaap ctpacccgaa aatcatcctg tetcgtcaga aacqtgacac cacccgtaac egtaacgact actctgacga atctgttatc ctggaaacca tcaacttcg tcgttacgct gctaccgctt ctgctgttct gCtggttet accgttaaag ttatcgttaa cgttgaaggt gaccegtcta tcgctgctaa cgaatcato cgttctatga aatag SEQ ID NO: 28 atgatgggt accttgtctt totaggacct ccaggtpcag gaaaaggaac ctacgcaaag agattgcagg aaataacpgg gattcctcat atatccaccg gtgacatttt cagggacatt gtaaaaaaag agaacgacga gcttgggaaa aagataaaag agatcatgga aaggggagaa etcgttccgg acgaactcgt gaacgaggtt gtgaaaagaa gactetcaga aaaagattgt gaaagagat tcatactgga eggotatcca apaaccgttg ctcappogga attectogac gpctttttga aaactcaaaa caaagetc acggctgctg tactctttga apttcetgag gaagtggtcg ttcagagct cacggccaga aggatetcc cgaaatgtgg aagaatttac aatttgattt cgctccctcc aaaagaagac paactgtgcg atgattgtaa agtgaagetc gttcagagag aagacpacaa agaagaaaca gtgagacaca WO 2010/079357 PCT/GB2010/050018 54 gatacaaggt ttatctcgaa aagacacage cagtqattga ttactacgat aaaaagggca ttctcaaacg agtggatggt accataggaa tagacaacgt gatcgctgaa gtgttaa aga taatagggtg gtgataaa tga SEQ ID NO: 29 attgatgc:ct attctggtttt tettggtcca ccgggggcag gcaaaggiac atatgcgaaa cgtttacagg aaatca;c:cgg czatc:ccgcac attagcacqg gcgacattt tcgtgqatatt gtcaaaaagg aaaatgacga attaggtaag aaaattaaag aaattatgga gegcggcgag ttggtgccgg acgaaeItggt gaatgaagtt gtcaaacgtc ggetc:tga aaaggattgc gaacgtgget ttattttgga cggttacc:cg cqtacagtag ctcaqgcaqa gtttctcqac ggcttcctqa agactcagaa taaggagtta acggegegg tcctgttcga ggtgcctgaa 9aggtggtcg ttcagcgtet gaccgcgegg cgttctgc:c cgaagtgtgg tcgtatttac aacctgattt cacttcctcc aaaagaagat gaactgtgtg atgactgcaa agtaaaactg gtgcaacgcgl aagatgataa agaggaaact gctg9Cgc.ZCt gctacaaagt atatctggaa aaaacccaac cgqgttatcga ttattatgat aaaaaaggca ttttgaaacg cgttgatgqg accatcggca tcgataacgt gattgcegaa gttctcaaaa tcattgggtg gagtgataaa SEQ ID NO: 30 atgaacctga ttttcctggg tccgcctggg gcaggcaaag gcacocagge gaaacgtgtg tctgaaaagt acggtatccc gcagattagt accggcqata tgotgeqtga agcggttgct aagggtacgg aactggqgaa aaaggegaaa gaatatatgg acaaagqgga acttgttccg gatgaagtag ttattggaat cgtgaaagaa cgcctc:cajgc aaccggattg tgagaagggc titattctgg ac:ggttttcc gcgtacgtta gcacaaqgccg aagctctgga cgaaatgtta aaagaattga ataagaaaat tgacgccgta atcaacgtgg tqtaccgga agaggaagtt gtcaagcgta ttacctatcg tcgcacttgc cgcaattgcg gcgc:ggtta ccatctcatt tatgcacctc caaaagagga taataaatgt gataaatgcq qcggtgagct ttatcagogt gatgacgata aagaaqagac agtccqgaq cgttaccgtg tgtataaaca gaa1c:acagag ccattgatcg attattaccg taaaaaggga at:ctgtatg atgtggatgg tactaaagac altcgaaggag tttggaaaga aattgaggeg attctggaaa aaattaaaag c SEQ ID NO: 31 Met Lys lie Gly Ile Val Thr GTy lie Pro Gly Val Gly Lys Ser Thr Val Leu Ala Lys Val Lys Gu lie Leu Asp Asn Gin Giy Ile Asn Asn Lys le lie Asr Tyr Gly Asp Phe Met Leu Ala Thr Ala Leu Lys Leu Giy Tyr Ala Lys Asp Arg Asp Gu Met Arg LVs Leu Ser Val Glu Lys Gin Lys Lys Leu Gn lie Asp Ala Ala Lys Gv Ile Ala Gu Glu Ala Arg Ala Gly Gly Giu Gly Tyr Leu Phe le Asp Thr His Ala Val lie Arg Thr Pro Ser Gly Tyr Leu Pro Gly Leu Pro Ser Tyr Val lie Thr Giu Ile Asn Pro Ser Val lie Phe Leu Leu Gu Ala Asp Pro Lys ie lie Leu Ser Arg Gin Lys Arg Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp GIu Ser VaI le Leu Gu Thr lie Asn Phe AL Arg Tyr Ala Ala Thr Ala Ser Ala Val Leu Ala Gly Ser Thr Val Lys Val lie Val Asn Val Giu Gly Asp Pro Ser lie Ala Ala Asn Gu lie ile Arg Ser Met Lys SEQ ID NO: 32 Met Met Ala Tyr Leu Val Phe Leu Gy Pro Pro Gly Ala Gly Lys Giv Thr Tyr Ala Lys Arg Leu Gin Glu lie Thr Gly Ile Pro His ie Ser Thr Gly Asp lie Phe Arg Asp Ie Val Lys Lys Giu Asn Asp GWu Leu (y Lys Lys liE L ys G1u le Met Gu Arg Gly Gu Leu Val Pro Asp Glu Leu Val Asn Glu Val Val Lys Arg Arg Leu Ser Giu WO 2010/079357 PCT/GB2010/050018 55 Lys Asp Cys Giu Arg Gly Phe Ile Leu Asp Gly Tyr Pro Arg Thr Val Aa GIn Aa Glu Phe Leu Asp Gly Phe Leu Lvs Thr Gin Asn Lys Glu Leu Thr Ala Ala Val Leu Phe Glu Val Pro Glu Gu Val Val Val Gin Arg Leu Thr Ala Arg Arg lie Cys Pro Lys Cys Gly Arg lie Tyr Asn Leu lie Ser Leu Pro Pro Lys Giu Asp Glu Leu Cys Asp Asp Cys Lys Val Lys Leu Val GIn Arg Gu Asp Asp Lys Giu Gu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Giu Lys Thr Gin Pro Val lie Asp Tyr Tyr Asp Lys Lys (ly lie Leu Lys Arg Val Asp Gly Thr lie Gly lie Asp Asn Val lie Ala Glu Val Leu Lys lie lie Gly Trp Ser Asp Lys SEQ ID NO: 33 Met Asn Gin Giu Gin Val Ser Pro Leu Gly Gly SEQ ID NO: 34 Met Asn Gin Gu Gin Val Ser Pro Leu Gly Gly Lys Ile Gly lie Val Thr Gly lie Pro Gly /al Gly Lys Ser Thr Val Leu Ala Lys Val Lys Glu Ile Leu Asp Asn GIn Gly lie Asn Asn Lys lie lie Asn Tyr Gly Asp Phe Met Leu Ala Thr Ala Leu Lys Leu Gly Tyr Ala Lys Asp Arg Asp Glu Met Arg Lys Leu Ser Val Glu Lys Gin Lys Lys Leu Gin Ile Asp Ala Ala Lvs GKy lIe Ala Giu Glu Ala Arg Ala Gy GSly Giu Gly Tyr Leu Phe lie Asp Thr His Ala Va lie Arg Thr Pro Ser Gly Tyr Leu Pro Gly Leu Pro Ser Tyr Val lie Thr Glu lie Asn Pro Ser Val lie Phe Leu Leu Gu AL Asp Pro Lys lie Ile Leu Ser Arg Gin Lys Arg Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp Glu Ser Val lie Leu Glu Thr lie Asn Phe Ala Arg Tyr Ala Ala Thr Ala Ser Ala Val Leu Ala Giy Ser Thr Val Lys Val lie Val Asn Val Giu Gly Asp Pro Ser lie Ala Ala Asn Glu lie Ile Arg Ser Met Lys SEQ ID NO: 35 Met Lys lie Gly lIe Val Thr Gly lie Pro Gly Val Gly Lys Ser Thr Val Leu Ala Lys Val Lys Glu lie Leu Asp Asn GIn Gy lIe Asn Asn Lys lie lie Asn Tyr Gly Asp Phe Met Leu Ala Thr Ala Leu Lys Leu Gly Tyr Ala Lys Asp Arg Asp Giu Met Arg Lys Leu Ser Val Giu Lys Gin Lys Lys Leu Gin lie Asp Ala Ala Lys Gly lie Ala GIU Glu Ala Arg Ala Gy Giv Glu Gly Tyr Leu Phe lie Asp Thr His Ala Val lie Arg Thr Pro Ser Gly Tyr Leu Pro Glv Leu Pro Ser Tyr Val lie Thr Glu lie Asn Pro Ser Val Ile Phe Leu Leu Glu Ala Asp Pro Lys lie iie Leu Ser Arg Gin Lys Arg Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp Glu Ser Val le Leu Glu Thr lie Asn Phe Ala Arg Tyr Ala Ala Thr Ala Ser Ala Val Leu Ala Gly Ser Thr Val Lys Val lie Val AsnVal GIu Gly Asp Pro Ser lie Ala Ala Asn Glu lie lie Arg Ser Met Lys Gly Gly Asn GIn GIu Gin Val Ser Pro Leu SEQ ID NO: 36 Met Asn Gin Glu Gin Val Ser Pro Leu Gly Gly Lys ile Gly lie Val Thr Gly lie Pro Gly Val Gly Lys Ser Thr Val Leu Ala Lys Val Lvs Glu lie Leu Asp Asn GIn Gly lie Asn Asn Lys lie lie Asn Tyr Gly Asp Phe Met Leu Ala Thr Ala Leu Lys Leu Gly Tyr Ala Lys Asp Arqg Asp Glu Met Arg Lys Leu Ser Val Glu Lys Gin Lys Lys Leu Gin lie Asp Ala Ala Lys GlV Ile Ala Gu Glu Ala Arg Ala Gy Giy Gu Gly Tyr Leu Phe lie Asp Thr His ALa Val lie Arg Thr Pro Ser Gly Tyr Leu Pro Gly Leu Pro Ser Tyr Val lie Thr Glu lie Asn Pro Ser Va lie Phe Leu Leu Glu Ala Asp Pro Lys lie lie Leu Ser Arg Gin Lys Arg Asp Thr Thr Arg Asn Arg Asn Asp TVr Ser WO 2010/079357 PCT/GB2010/050018 56 Asp Giu Ser Val lie Leu Giu Thr lIe Asn Phe Ala Arg Tyr Ala Ala Thr Ala Ser Ala Val Lou Ala Gly Ser Thr Val Lys Val lie Val Asn Val Glu Gly Asp Pro Ser lie Ala Ala Asn Gu lie lie Arg Ser Met Lys Gly Gly Asn Gin Gu Gin Val Ser Pro Lou SEQ ID NO: 37 atgaatcaag aacaagtcaq ccqotgggc qgcatcatcg cctatctggt ttttcttggt ccaccggqgg caggcaaaqg tacctatqcg aaacgtttac aggaaatca cggcatccg cacattagca cgggcgacat ttttcgtgat attgtcaaaa aggaaaatga cgaattaggt aaqaaaatta aagaaattat ggagcgogg gagttqgtqc eggacgaact qtQaatqaa gttgtcaaac gtcggctgtc tgaaaaggat tgcgaacgtg geitttatit ggacggttac ccgcgtacag tagotcagg oagtttctc gacggcttcc tgaagactca gaataaggag ttaacggctg cggtcctgtt cgaggtgcct gaagaggtgg tcgttcagcg tctgaccgcg cggcgtatct gccgaagtg tggtcgtatt tacaacctga tttcacttcc tccaaaagaa gatgaactgt gtgatgactg caaagtaaaa ctggtgcaac gcgaagatga taaagaggaa actgtgcglcc atcgctaca agtatatctg gaaaaaccc aaccggttat ogattattat gataaaag gcattttgaa acgcgttgat gggaccatcg gcatcgataa cgtgattgcc gaag ttctca aaatcattgg gtggagtgat aaatggtg acgc: SEQ ID NO: 38 Met Asn GIn Gu Gin Val Ser Pro Leu Gly Gy lie lie Ala Tyr Lou Val Phe Leu Gy Pro Pro (Ty Ala Gly L s Gy Th Tyr Ala Lys Arg Leu GIn GTu He Thr Gly lIe Pro 1Hs He Ser Thr Gly Asp He Phe Arg Asp le Val Lys Lys Gu Asn Asp Giu Lou (Ty Lys Lys lIe Lys Gu lie Met (u Arg (y GTu Lou Vol Pro Asp GIu Lou Val Asn GIu Val Val Lys Arg Arg Leu Ser Gu Lys Asp Cys GIu Ag Gly Phe lIe Leu Asp Gy Tyr Pro Arg Thr Val Ala Gin Ala Gu Phe Leu Asp Gly Phe Leu Lys Thr GTn Asn Lys GTu Lou Thr Ala Ala Val Leu Phe Gu Val Pro Glu Gu Val Val Val Gin Arg Leu Thr Ala Arg Arg le Cys Pro Lys Cys Gly Arg lIe Tyr Asn Leu le Ser Lou Pro Pro Lys GTu Asp Gu Lou Cys Asp Asp Cys Lys Val Lys Lou Val Gin Arg Glu Asp Asp Lys Glu Glu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Gu Lys Thr Gin Pro Val lie Asp Tyr Tyr Asp Lys Lys Gly 1He Lou Lys Ag Val Asp (Ty Thr lIe Gy le Asp Asn Val lIe Ala Giu Val Leu Lys lIe lbe Gy Trp Ser Asp Lys SEQ ID NO: 39 atgatggcct atctggtttt tcttggtcca ccgggggcag gcaaaggtac ctatgcgaaa cgtttacagg aatcaccgg catcccgoc attagcacgg gcgacatttt tcgtgatatt gtcaaaaagg aaaatgacga attaggtaag aaaattaaag aaattatgga gegoggogag ttggtgccgg acgaactggt gaatgaagtt gtcaaacgtc ggctgtxtga aaaggattgc gaacgtggct ttattttgga cggttacccg cgtacaqtag ctcaggcaga gtttctcgac qgcttcctga agactcagaa taaggagtta acgqctgcgq tcctqttcga ggtgcctgaa gaggiggtcg ttcagcgtct gaccgcgcgg cgtatctgcc cgaagtgtgg tcgtatttac aacctgattt cacttcctcc oaaagaagat gaactgtgtg atgactgcaa agtaaaactg gtgcaacgcg aagatgataa agaggaaact gtgcgccatc gctacaaagt atatctggaa aaaacccaac cggttatcga ttattatgat aaaaaggca ttttgaaacg cgttgatggg accatcggca tcgataacgt gattgccgaa gttctcaaaa tcattgggtg gagtgataaa ctgggcggc atcgaaca agtcagcccg ctgta SEQ ID NO: 40 WO 2010/079357 PCT/GB2010/050018 57 Met Met Ala Tyr Leu Val Phe Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Tyr Ala Lys Arg Leu Gin Giu lie Thr GIV Ile Pro His lIe Ser Thr GLy Asp lie Phe Arg Asp lIe Val Lys Lys Glu Asn Asp Gu Leu Gly Lys Lys lie Lys Gu lie Met Glu Arg Giy GIU Leu Val Pro Asp Glu Leu Val Asn GIu Val Val Lys Arg Arg Leu Ser Glu Lys Asp Cys Glu Arg Gly Phe le Leu Asp Gly Tyr Pro Arg Thr Vai Ala Gin Ala Glu Phe Leu Asp Giy Phe Leu Lys Thr Gin Asn Lys Gu Leu Thr Ala Ala Val Leu Phe GIu Val Pro Glu Gu Val Val Val Girt Arg Leu Thr Aia Arg Arg le Cys Pro Lys Cys Gly Arg le Tyr Asn Leu le Ser Leu Pro Pro Lys Gu Asp Glu Leu Cys Asp Asp Cys Lys Val Lys Leu Val Gin Arg Giu Asp Asp Lys Gu Giu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Glu Lys Thr Gin Pro Val lie Asp TVr Tyr Asp Lys Lys Gly lie Leu Lys Arg Val Asp Gly Thr lie Gly Ile Asp Asn Val lie Ala Giu Val Leu Lys lie le Gly Trp Ser Asp Lys Leu Gly Gly Asn Gin Gu Gin Val Ser Pro Leu SEQ ID NO: 41 atgaatcaag aacaagtcag c01cgtggc ggcatcatcg cctatctggt ttcttggt ccaccggggg caggcaaagg tacctalgeg aaacgttrac aggaaatcac cggcjatcccg cacattagca cgggcgacat ttttcgtgat attgtcaaaa aggaaaatga cgaattagqt aagiaaaatta aagaaattat ggagjcgcggC gagttggtgc cggacgaact ggtgaatgaa gttgtcaaac gtcggtgtc: tgaaaaggat tgcgaacgtg qctttatttt ggacggttac ccgcqtacag tagotcaggo agagittctc gacggcttcc tgaagactca gaataaggag tIaacggctg cggtc:ctgtt cgaggtgcct gaagaggtg tcgttcagcg tctgaccgc:g cggcgtatct gcccgaagtg 1tggtcgtatt tacaacctga ttcactc tocaaaagaa gatgaactgt gtgatgactg caaagtaaaa ctggtgcaac qcgaagatga taaagaqgaa actgtgcgcc atc:gctacaa agltatatctg gaaaaaac:cc aaccggttat cgattattat gataaaaaag gcattttgaa acgcgttgat gggaccatcg gcatcgataa cgtgattgcc gaagttca aaatcattgg gtggagtgat aaactgggcg gcaatcaaga acaagtcage eccctgtaa SEQ ID NO: 42 Met Asn Gin Gu Gin Val Ser Pro Leu Gly Gly lie lie Ala Tyr Leu Val Phe Leu (ly Pro Pro Gly Ala Gly Lys Gly Thr Tyr AL Lys Arg Leu Gin GIu lie Thr Gly lie Pro His lie Ser Thr Gly Asp lie Phe Arg Asp lie Val Lys Lys Glu Asn Asp Glu Leu Gly Lys Lys lie Lys Giu lie Met Giu Arg Gly Glu Leu Val Pro Asp GIu Leu Val Asn Glu Val Val Lys Arg Arg Leu Ser Gu Lys Asp Cys Gu Argp Gy Phe lie Leu Asp Gly Tyr Pro Arg Thr Val Ala Gin Ala GIu Phe Leu Asp Gly Phe Leu Lys Thr Gin Asn Lys Giu Leu Thr Ala Ala Val Leu Phe Giu Val Pro Giu Gu Val Val Val Gin Arg Leu Thr Ala Arg Arg lie Cys Pro Lys Cys Gly Arg Ile Tyr Asn Leu lie Ser Leu Pro Pro Lys Gu Asp Glu Leu Cys Asp Asp Cys Lys Val Lys Leu Val Gin Arg Glu Asp Asp Lys Glu Glu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Gu Lys Thr Gin Pro Val lie Asp Tyr Tyr Asp Lys Lys Gly lie Leu Lys Arg Val Asp Giy Thr lie Gly lie Asp Asn Va lie Ala Giu Val Leu Lys lie le Gly Trp Ser Asp Lys Leu Gly Gly Asn GIn Gu Gin Val Ser Pro Leu SEQ ID NO: 43 gattcaaacc aaggcaacaa tcagcaaaac taccagcaat acagccagaa cggtaaccaa caacaaggta acaacagata ocaaggttat caagcttaca atgctcaagc ceaacctggg ggtgggtact accaaaatta ccaaggttat tctgggtacc aacaaggtgg ctatcaacag tacaatcccg acgccggtta ccagcaacag tataatcctc aaggaggcta tcaacagtac aatcctcaag gcggttatca WO 2010/079357 PCT/GB2010/050018 58 gcaccaatto aatccacaag gtggccgtgg aaattacaaa aacttcaact acaataacaa tttgcaagga tatcaagctg gtttccaacc acagtctcaa ggtatgtctt tgaacgactt tcaaaagcaa caaaagcagg cegetcccaa accaaagaag actttgaage ttgiteccag ttectgtatc aagttgqcca atgotacca gaagqgttgac acaaaacctg ccgaatctg taagaaagag rgaaga t ctgctgaaac caaagaacca actaaagage caacaaaggt cgaagaacca gtt;laaaaagg aggagaaacc agtccagact gaagaaaaga cggaggaaaa atcggaactt ccaaaggtag aagaccttaa aatctctgaa tcaacacata ataccaacaa tgccaatgtt accagtgctg atg:ccttgat caaggaacag gaagaagaag tggatgacga agttgttaac gatatgtttg gtggtaaaga tcacgtttct ttaattttca tgggtatgt tgatgccggt aaatctacta tgggtjgtaa tctactatac ttgactggct ctgtggataa gagaactatt gagaaatatg aaagagaagc caaggatqca qgcaqacaag gttigtactt gtcatggqtc atqgatacca acaaagaaga aagaaatgat ggtaagacta tcgaagttgg taaggcctac tttgaaactg aaaaaaggcg ttataccata ttggatgctc ctggtcataa aatgtacqtt tcogagatga tcggtggtgc tttcaaget gatgitggtg ttttggtcat ttcogccaga aagggtgagt acgaaaccgg ttttgagaga ggtggtcaaa etegtgaaca cgccctattg gccaagaccc aaggtgttaa taagatg gt::gtcgtaa ataagatgga tgacccaacc gttaactggt ctaaggaacg ttacgaccaa tgtgtgagta atgtcagcaa tticttga SEQ ID NO: 44 Asp Ser Asn Gin Gly Asn Asn Gin (Gn Asn Tyr Gin Gin Tyr Ser Gin Asn Gly Asn GIn Gin Gin GIy Asn Asn Arg Tyr Gin Gly Tyr GIn Ala Tyr Asn Ala Gln Ala GIn Pro Gly Gly Gly Tyr Tyr GIn Asn Tyr Gln Gly Tyr 3er Gly Tyr Gin Gin Gly Gly Tyr GIn Gin Tyr Asn Pro Asp Ala Gly Tyr Gin (Gn Gin Tyr Asn Pro (Gn Gy Gly Tyr Gin Gn Tvr Asn Pro GIn Gly Gly Tyr GIn His GIn Phe An Pro GIn Gy Glv Arg Gy Asn Tyr Lye Asn Phe Asn Tyr Asn Asn Asn Leu Gin Gly Tyr Gin Ala Gly Phe GIn Pro Gin Ser Gin Gy Met Ser Leu Asn Asp Phe GIn Lys Gin Gin Lys Gin Ala Ala Pro Lys Pro Lys Lys Thr Leu Lys Leu Val Ser Ser SEr Cys lIe Lys Leu Ala Asn Ala Thr Lys Lys Val Asp Thr Lys Pro Ala Gu Ser Asp Lys Lys Gu Glu Gu Lys Ser Ala Glu Thr Lys Gu Pro Thr Lys Gu Pro Thr Lys Val Gu Gu Pro Val Lys Lys GIu Gu Lys Pro Val Gn Thr Gu Gu Lys Thr GIu Glu Lys Ser Gu Leu Pro Lys Val Gu Asp Leu Lys lie Ser Gu Ser Thr His Asn Thr Asn Asn Ala Asn Val Thr Ser Ala Asp Ala Leu lie Lys Gu GIn Gu Gu Gu Val Asp Asp Gu /al Val Asn Asp Met Phe Gly Gly Lys Asp lis Val Ser Leu lIe Phe Met Gly His Val Asp Ala Gly Lys Ser Thr Met Gly Gy Asn Leu Leu Tyr Leu Thr Gly Ser Val Asp Lys Arg Thr lIe Gu Lys Tyr Gu Arg Glu Ala Lys Asp Ala Gly Arg Gin Gly Trp Tyr Leu Ser Trp Val Met Asp Thr Asn Lys GIu Giu Arg Asn Asp Gly Lys Thr lIe (GIu Val Gly Lys Ala Tyr Phe Gu Thr Glu Lys Arg Arg Tyr Thr lIe Leu Asp Ala Pro Gly His Lys Met Tyr Val Ser Glu Met Ile Gly Gly Ala Ser GIn Ala Asp Val Gly Val Leu Val lIe Ser Ala Arc Lys Gly Glu Tyr Giu Thr Gy Phe Gu Arg Gly Gly GIn Thr Arg GIu His Ala Leu Leu Ala Lys Thr Gin Gly Val Asn Lys Met Val Val Val Val Asn Lys Met Asp Asp Pro Thr Val Asn Trp Ser Ly Glu Arg Tyr Asp Gin Cys Val Ser Asn Val Ser Asn Phe Leu SEQ ID NO: 45 atggactcta accagggtaa caaccagcag aactaccagc agtactctca gaacggtaac cagcagcagg gtaacaaccg ttaccagggt taccaggctt acaacgctca ggctcagceg ggtggtggtt actaccagaa ctaccagggt tactccggat atcaacaggg tggttaccaa caatataatc cagacgctgg ttaccagcag cagtacaacc cgcagggtgg ttaccagcag tacaacccgc aaggcggata tcaacaccag ttcaatccgc agggtggtcg tggtaactac aaaaacttca actacaacaa caacctgcag ggttaccagg ctggttaa WO 2010/079357 PCT/GB2010/050018 59 SEQ ID NO: 46 Met Asp Ser Asn Gin Giv Asn Asn Gin Gin Asn Tyr Gin Gin Tyr Ser Gin Asn Gly Asn Gin Gin Gin Gly Asn Asn Arg Tyr Gin Gly Tyr Gin Ala Tyr Asn Ala Gin Ala Gin Pro Gly Gly Gly Tyr Tyr Gin Asn Tyr Gin Gly Tyr Ser Gly Tyr Gin Gin Gly Gly Tyr Gin Gin Tyr Asn Pro Asp Ala Gly Tyr Gin Gin Gin Tyr Asn Pro Gin Gly Gly Tyr Gin Gin Tyr Asn Pro Gin Gly Gly Tyr Gin His GIn Phe Asn Pro Gin Gly Gly Arq Gly Asn Tyr Lys Asn Phe Asn Tyr Asn Asn Asn Leu Gin Giy Tyr Gin Ala Gly SEQ ID NO: 47 atgqactcta accagggtaa caaccagcag aactaccagc agtactctca gaacgqtaac cagcagcagg gtaacaaccg ttaccagggt taccaggctt acaacgctca ggctcagccg ggtggtggtt iactaccagaa ctaccagggt tactccggtt atcagcaagg tggctaccaa caatataatc cagacgctgg ctatcaacag caatataatc otcagggtgg ttaccaqcag tacaacccgc aagqcggtta tcaacaccag ttcaatccgc agggtggtog tggtaactac aaaaactica actacaacaa caacctgcag ggttaccagg ctggaatt gaagatcqgc attgtgaccg gcattocggg cgttqgcaaa agcaccgttc tggcaaagqt gaaggagatc ctggacaaccaqggcattaa taacaaaatt attaattatg gtgattttat gctggcgacc gcgctgaagc tggge:tacgc: aaaagatcgt gacgaaatgc gcaaactgag cgtgqaaaaa cagaagaage Igcagattga tgeggcgaaq ggcattgcgg aagaggcaccg cqcgggcggc gaagqctacc tgtttatcga ta;ccca tgcg gtgatccgca ccccgagcgg ttatctgocg ggcc:tgccgt cttacgtgat tacgg -aaatc: aacccgag:g ttatttttt gctggagqca gatcogaaga ttattctgag ccqccagaag cgcgatacca cccgcaaccg caacgattat agcgacgaaa gcgttatcct ggagaccate aactttgcgc gctatgcggc aaccgcgagc gcggttctgg caggctctac cgttaaagtg alegtgaacg tggaggqtga tccaagcatc gcgqcgaacg aaatcattog cagcatgaaa taagicqacg c SEQ ID NO: 48 Met Asp Ser Asn Gin Gly Asn Asn Gin Gin Asn Tyr GIn Gin Tyr Ser GIn Asn Gly Asn Gin Gin Gin GlyAsn Asn Arg Tyr Gin Gly Tyr Gin Ala Tyr Asn Ala Gin Ala Gin Pro Gly Gly Gly Tyr Tyr Gin Asn Tyr Gin Gly Tyr Ser Gly Tyr Gin Gi Gly Gly Tyr Gin GIn Tyr Asn Pro Asp Ala G' Tyr Gin Gi Gin Tyr Asi Pro Gin Gly Gly Tyr Gin Gin Tyr Asn Pro Gin Gly Gly Tyr Gin His GIn Phe Asn Pro Gin Gly Gly Arq Gly Asn Tyr Lys Asn Phe Asn Tyr Asn Asn Asn Leu Gin Glv Tyr Gin Ala Gly Ile Met Lys lie Gly lie Val Thr Gly lie Pro GOy Val Giv Lvs Ser Thr Val Leu Ala Lys Val Lys Gu lie Leu Asp Asn Gin Gly lie Asn Asn Lys lie lie Asn TVr GIy Asp Phe Met Leu Ala Thr Ala Lieu Lys Lou GlV Tyr Ala Lys Asp Arg Asp Giu Met Arg Lys Leu Ser Val Gu Lys Gin Lys Lys Leu Gin ile Asp Ala Ala Lys Gly lie Ala Giu GILu Ala Arg Ala Gly Gly Gliu Gly Tyr Leu Phe lie Asp Thr His Ala Val lie Arg Thr Pro Ser Gly Tyr Leu Pro Gly Leu Pro Ser Tyr Val Ile Thr Giu lie Asn Pro Ser Val Ile Phe Leu Leu Giu Ala Asp Pro Lys lie ie Leu Ser Arg Gin Lys Arqg Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp Glu Ser Vai lie Leu Glu Thr Ile Asn Phe Ala Arg Tr Aia Ala Thr Ala Ser Ala Val Leu Ala Giv Ser Thr Val Lys Val lIe Val Asn Val Gu Gly Asp Pro Ser lie Ala Ala Asn Giu lIe ile Arg 5er Met Lys SEQ ID NO: 49 atgaagatcg gcattgtgac cggcattccg ggcgttggca aaagcaccgt tctggcaaag gtgaaggaga tcctggacaa ccagggcatt aataacaaaa ttattaatta tggtgatttt atgctggcga cucgctgaa gctgggetac gcaaaagatc gtgacgaaat gcgcaaactg WO 2010/079357 PCT/GB2010/050018 60 agcgtggaaa aacagaagaa gctgcagatt gatgcggcga agggcattgc ggaagaggca cgqggggcg gegaaggcta cctgtttatc gatacccatg cggtgatccg caccccgage ggttatctgc cgggcctgcc gtcttagtg attacggaaa tcaacccgag cgttatttti ctgctggagg cagatccgaa gatattctg agcegccaga agegegatac cacccgcaac cgcaacgatt atagegacqa aagcgttatc ctggagacca tcaactttgc gcgctatgcg gcaaccgCga gcceggtte t ggcaggtcet accgttaaag tgatcgtgaa cgtggagqgt gatccaagca tcgcggcgaa cgaaatcatt cgcagcatga aacagtegag tatggactct aaccagggta acaaccagca gaactaccag cagiac;tctc agaacggtaa ccagcagcag ggtaacaacc gitaccaggg ttaccagget tacaacgctc aggetc:agcc gggtggtggt tactaccaga actaccaggg ttactccggt tatcagcaag gtggctacca acaatataat ccagacqtg gctatcaaca gcaatataat cctcagggtg gttaccagca gtacaacccg caaqggcggtt atcaacacca gttcaatccg cagggtggte gtggtaacta caaaaactto aactacaaca acaacctgca gggttaccag gctggttaag tcgacgc SEQ ID NO: 50 Met Lys lie GCy lie Val Thr Gly Ile Pro Gly Val Glv Lys Ser Thr Val Leu Ala Lys Val Lvs Glu lie Leu Asp Asn Gin Gly lie Asn Asn Lvs lie lie Asn Tyr Gly Asp Phe Met Leu AL Thr Ala Leu Lys Leu Gly Tyr Ala Lys Asp Arq Asp Glu Met Arg Lys Leu Ser Val Giu Lys Gin Ls Lys Leu Gin lie Asp Ala Ala Lys Gly Ile Ala Giu Giu AL Arg Ala Gly Gly GIu Gly Tyr Leu Phe lie Asp Thr His Ala Val lie Arg Thr Pro Ser Gly Tyr Leu Pro Gy Leu Pro Ser Tyr Val lIe Thr Glu ile Asn Pro Ser Va lie Phe Leu Leu Giu Ala Asp Pro Lys lie lie Leu Ser Arg Gin Lys Arg Asp Thr Thr Arg Asn Arg Asn Asp T yr Ser Asp Gu Ser Val Ile Leu Gu Thr lie Asn Phe Ala Arg Tyr Ala Ala Thr Ala Ser Ala Val Leu Ala Gly Ser Thr Val Lys Val lie Val Asn /al Glu Gly Asp Pro Ser lie Ala AL Asn Glu lie lie Arg Ser Met Lys Gin Ser Ser Met Asp Ser Asn Gin Gly Asn Asn Gin Gin Asn Tyr Gin Gin Tyr Ser Gin Asn Gly Asn Gin Gin Gin Gly Asn Asn Arg Tyr Gin Gly Tyr Gin Ala Tyr Asn Ala Gin Ala Gin Pro Gly Gly Gly Tyr Tv GIn Asn Tyr Gin Gly Tyr Ser Gly Tyr Gin GIn Gly Gly Tyr Gin Gin Tyr Asn Pro Asp Ala Gly Tyr GIn Gin Gin Tyr Asn Pro GIn Gly Gly Tyr Gin Gin Tyr Asn Pro Gin Gly Gly Tyr Gn His Gin Phe Asn Pro Gin Gly Gv Arg Gly Asn Tyr Lys Asn Phe Asn Tyr Asn Asn Asn Leu Gin Gly Tyr Gin Ala Gly SEQ ID NO: 51 atgjactcta accagggtaa caaccagcag aactaccage agtactctca gaacggtaac cagcagcagg gtaacaaccg ttaccagggt taccaqggit acaacgctca ggctcagccg ggtggtggtt actaccagaa ctaccagggt tactccggt atcagcaagg tggctaccaa caatataate cagacgctgg ctatcaacag caatataatc ctcagggtgg ttaccaceag tacaacccgc aaggcggtta tcaacaccag ttcaatccgc agggtgtcg tggtaactac aaaaacttca actacaacaa caacetgcag ggttaccagg ctggaattat gatggcctat ctggtttttc ttggtccacc gggggcaggc aaaggacct atgcgaaacg tttacaggaa atcaccggca tccogcaat tagcacggc gacattttic qtgatattqt caaaaaggaa aatgacgaat taggtaagaa aattaaagaa attatggagc gcggcgagtt ggtgcC:9gga: ga:actggtga atgaagttgt caaacgtcgg ctgictgaaa aggattgc:ga acgtggcttt attttggacg gttacccgcg tacaqgtagct caggcagagt tctcgacgg cttectgaag actcagaata aggagttaac ggctgcggtc ctgttcgagg tgcctgaaga ggtggctgtt cagcgtetga ccggCggcg tatctgcecg aagtgiggtc gtatttacaa cctgatttca cttcctccaa aagaagata actgtgtgat qactgcaaag taaactggt gcaacqcgaa gatgataaag aggaaactgt gegccatoge tacaaagtat atctggaaaa aacccaaccg gttatcgatt attatgataa aaaaggcatt ttgaaacgcg ttgatggac catcggcatc gataacgta ttgccgaagt tctcaaaatc attggga gtgataaata g WO 2010/079357 PCT/GB2010/050018 61 SEQ ID NO: 52 Met Asp Ser Asn Gin Gly Asn Asn Gin GIn Asn Tyr GIn Gin Tyr Ser Gin Asn Gly Asn Gin Gin Gin Gy Asn Asn Arg Tyr Gin Gly Tyr GIn Ala Tyr Asn Ala GIn Ala Gin Pro Gly Gly Gly Tyr TyrGIn Asn Tyr Gin Gly Tyr Ser Gy Tyr Gin GIn Giy Giy Tyr Gin Gin Tyr Asn Pro Asp Ala Gly Tyr Gin Gin Gin Tyr Asn Pro GIn Giv Giv Tvr Gin Gin Tyr Asn Pro Gin GIy Giv Tyr Gin His GIn Phe Asn Pro Gin Gly Gly Arg Gly Asn Tyr Lys Asn Phe Asn Tyr Asn Asn Asn Leu Gin Gly Tvr Gin Ala Gly lIe Met Met ALa Tyr Leu Val Phe Leu Gly Pro Pro Gy Ala Gly Lvs Gly Thr TVr Ala Lys Arg Leu Gin Glu lie Thr Gly lie Pro His lie Ser Thr Gly Asp lie Phe Arg Asp ie Val Lys Lys Giu Asn Asp Giu Leu Gly Lys Lys lie Lys Giu ie Met Giu Arg Glv Giu Leu Val Pro Asp Giu Leu Val Asn Glu Val Val Lys Arg Arg Leu Ser Glu Lys Asp Cys Glu Arg Gly Phe lie Leu Asp GlV Tyr Pro Arg Thr Val Ala Gin Ala Giu Phe Leu Asp Gly Phe Leu Lys Thr Grin Asn Lys Glu Leu Thr ALa Ala Val Leu Phe Glu Val Pro Giu Giu Val Val Val Gin Arg Leu Thr Ala Arg Arg Ile Cys Pro Lys Cys Gly Arg le Tyr Asn Le lie Ser Leu Pro Pro Lys Giu Asp Glu Leu Cys Asp Asp Cys Lys Val Lys Leu Val GIn Arg Giu Asp Asp Lys Glu Gu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Giu Lys Thr GIn Pro Va lie Asp Tyr Tyr Asp Lys Lys Gly lie Leu Lys Arg Val Asp Gly Thr lie Gly lie Asp Asn Vai lie Ala Giu Val Leu Lys lie lie Gly Trp Ser Asp Lys SEQ ID NO: 53 Ala Thr Gly Ala Thr GIy Gly Cys Cy T Aa Thr Gys Thr Giy Gy Thr Thr Thr Thr Thr Cys Thr Thr Giy Gy Thr Cys Cys Ala Cys Cys Gly Gly Gly Gly Gly Cys Ala Gly Gly Cys Ala Ala Ala Gly (Thy Tihr Ala Cys Cvs Thr Ala Thr Gly Cys Gly Ala Ala Ala Cys Gly Thr Thr Thr Ala Cvs Ala Gly Gly Ala Ala Ala Thr Cys Ala Cvs Cvs GI Giv Gys Ala Thr Cys Cys Cys Glv Cys Ala Cys Ala Thr Thr Ala Gly Cys Ala Cys Gly Gly Gly Cys Gly Ala Cvs AL Thr Thr Thr Thr Thr Cys Gly Thr Gly Ala Thr Ala *Thr Thr Gly Thr Cys Ala Ala Ala Ala Ala Giv Glv Ala Ala Ala Ala Thr Gly Ala Cvs Giv Ala Ala Thr Thr Ala Gly Gly Thr Ala Ala Gly Ala Ala Ala Ala hrT Thr Ala Ala Ala Giv Ala Ala Ala Thr Thr Ala Thr Gly Gly Ala Gly Cys Gly Cys Gly Gly Cys Gly Ala Gly Thr Thr Gly Gly Thr Gly Cys Cys Gly Gly Ala Cys Gly Ala Ala Cys Thr Gly Gly Thr Giy Ala Ala Thr Gly Ala Ala Giy Thr Thr Gly Thr Cys Ala Ala Ala Cys Gly Thr CSy Gly GTy s Thr Giy Thr Cys Thr Gly Ala Ala Ala Ala Gly Gly Ala Thr Thr Gby Cys Gly Ala Ala Cys Gly Thr Gy Gy Cys Thr Thr Thr Ala Thr Thr Thr Thr Gly Gly Ala Cys Gly Gly Thr Thr Ala Cys Cys Cys Gly Cys Gly Thr Ala Cys Ala Gly Thr Ala Giv Cys Thr Cys Ala Gly Gly Cys Ala Gly ALa Gly Thr Thr Thr Cys Thr Cys Gly Ala Cys Gly Gly Cys Thr Thr Cys Cys Thr Gly Ala Ala Gly Ala Cys Thr Cys Ala Gly Ala Ala Thr Ala Ala Gly Gly Ala Gly Thr Thr Ala Ala Cys Gly Gl, Cys Thr Gly Cys (Thy Gy Thr Cys Cys Thr Gly Thr Thr Cys Gly Ala Gly Gly Thr Gly Cys Cys Thr Gly Ala Ala Gly Ala Gly Gly Thr Gly Gly Thr Cys GlV Thr Thr Cys Ala Gly Cys Gly Thr Cys Thr Gly Ala Cys Cys Gy uys Ghy Cys Gly Gy Cys Gly Thr Ala Thr Cys Thr Gly Cys Cys Cys Gly Ala ALa Gly Thr Gly Thr Gly Gy Thr Cys Glv Thr Ala Thr Thr Thr Ala Cys Ala Ala Cys Cys Thr Giy Ala Thr Thr Thr Cys Ala Cys Thr Thr Cys Cys Thr Cys Cys Ala ALa Ala Ala Gy Ala Ala Gsy Ala Thr Gly Ala Ala Cys Thr Gly Thr Gly Thr Gly Ala Thr Gly Ala Cvs Thr Giy Cys Ala Ala Ala GlV Thr Ala Ala Ala Ala Cys Thr Glv Glv Thr Giv Cys Ala Ala Cys Gly Cys Gly Ala Ala Gky Ala Thr Gly Ala Thr Ala Ala Ala Gly Ala Gly Gly Ala Ala Ala Cys Thr Gly Thr Gly WO 2010/079357 PCT/GB2010/050018 62 Cys Giy Cys Cys Ala Thr Cys Giy Cys Thr Ala Cys Ala Ala Ala Gly Thr Ala Thr Ala Thr Cys Thr Gly Gly Ala Ala Ala Ala Ala Ala Cys Cys Cys Ala Ala Cvs Cys Gly Glv Thr Thr Ala Thr Cys Gly Ala Thr Thr Ala Thr Thr Ala Thr Gly Ala Thr Ala Ala Ala Ala Ala Ala Gly Gly Cys Ala Thr Thr Thr Thr Gly Ala Ala Ala Cys Gly Cys Gly Thr Thr Gly Ala Thr Gly Gly Gly Ala Cys Cys Ala Thr Cys Gly Gly Cys Ala Thr Cys Gly Ala Thr Ala Ala Cys Gly Thr Gly Ala Thr Thr Gly Cys Cys Giy Ala Ala Gly Thr Thr Cys Thr Cys Ala Ala Ala Ala Thr Cys Ala Thr Thr Giy GlyGly Thr Glv Gly Ala Gly Thr Gly Ala Thr Ala Ala Ala Cys Thr Gly Thr Cys Gly Ala Gly Thr Ala Thr Gly Gly Ala Cys Thr Cys Thr Ala Ala Cys Cys Ala -Gy -Gy Gly Thr Ala Ala Cys Ala Ala Cys Cys Ala Gly Cys Ala Gly Ala Ala Cys Thr Ala Cys Cys Ala Gly Cys Ala Gly Thr Ala Cys Thr Cys Thr Cys Ala Gly Ala Ala Cys Gly Gly Thr Ala Ala Cys Cys Ala Gly 'ys Ala Gly Cys Ala Gly Gly G 3 ly Thr Ala AL Cys AL Ala Cys Cys Gly Thr Thr Ala Cys Cys Ala GlV Glv Gly Thr Thr Ala Cys Cys Ala Gly Gly Cys Thr Thr Ala Cys Ala AL Cys Gly Cys Thr Cys Ala Gly Gly Cys Thr Cys Ala Gly Cys Cys Gly Gly Gly Thr Gly Gly Thr Glv Glv Thr Thr Ala Cys Thr Ala Cys Cys Ala Gly Ala Ala Cys Thr Ala Cys Cys Ala Gly Gly Gly Thr Thr Ala Cys Thr Cys Cys Gly Gly Thr Thr Ala Thr Cys Ala Gly Cys Ala Ala Gly Gly Thr Gly Gly Cys Thr Ala Cys Cvs Ala Ala Cys Ala Ala Thr Ala Thr Ala Ala Thr Cys Cys Ala Gly Ala Cys Gly Cys Thr Gly Gly Cys Thr Ala Thr Cvs Ala Ala Cys Ala Gk Cys Ala Ala Thr Ala Thr AL Ala Thr C's Cys Thr Cys Ala Gly Gly Gly Thr Gly Gly Thr Thr Ala Cys Cys Ala Gly Cys Ala Gly Thr Ala Cys Ala Ala CyVs Cys Cys Gly Cys Ala Ala Gy Gly Cys GKy Gly Thr Thr Ala Thr Cys Ala Ala Cys AL Cys Cys Ala Gly Thr Thr Cys Ala Ala Thr Cys Cys Gly Cys Ala Gly Gly Gly Thr Gly Gly Thr Cys Gly Thr Gly Gy Thr Ala Ala Cys Thr Ala Cys Ala Ala Ala Ala Ala Cvs Thr Thr Cys Ala Ala Cys Thr Ala Cys Ala Ala Cys Ala Ala Cys Ala Ala Cys Cys Thr GKy Cys Ala Gly Gly Gly Thr Thr Ala Cys Cys Ala Gly GIy Cys Thr GIy Gly Thr Thr Ala Ala Gly Thr Cys Gly Ala Cys Gly Cys SEQ ID NO: 54 Met Met Ala Tvr Leu Val Phe Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Tyr Ala Lys Arg Leu Gin Glu lle Thr GIv Ile Pro His lIe Ser Thr Gly Asp lie Phe Arg Asp lIe Val Lys Lys Giu Asn Asp Glu Leu Gly Lys Lys lIe Lys Glu lie Met Glu Arg Gly Giu Leu /al Pro Asp Giu Leu /al Asn Gu Val Val Lys Arg Arg Leu Ser Giu Lys Asp ;ys Glu Arg Gly Phe Ile Leu Asp Gly Tyr Pro Arg Thr Val AL GIn Ala Glu Phe Leu Asp Gly Phe Leu Lys Thr Gin Asn Lys Glu Leu Thr Ala Ala Val Leu Phe Giu Val Pro Glu Giu Val Val Val Gin Arg Leu Thr Ala Arg Arg lie Cys Pro Lys Cys Gly Arg Ile Tvr Asn Leu lie Ser Leu Pro Pro Lvs Glu Asp Gu Leu Cys Asp Asp Cys Lys Val Lys Leu Val Gin Arg Glu Asp Asp Lys Gu Giu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Gu Lys Thr GIn Prc Val lie Asp Tyr Tyr Asp Lys Lys Gly lie Leu Lys Arg Val Asp Gly Thr Ile Gly lie Asp Asn Val lie Ala Giu Val Leu Lys lie lie Gly Trp Ser Asp Lys Leu Ser Ser Met Asp Ser Asn Gin Gly Asn Asn GIn Gin Asn Tyr Gin Gin Tyr Ser Gin Asn Gly Asn Gin Gin Gin Ghy Asn Asn Arg Tvr Gin Gly Tyr GIn Ala Tyr Asn Ala Gn Ala GIn Pro Gly Gly Gly Tyr Tyr GIn Asn Tyr Gin Gly Tyr Ser Gly Tyr Gin Gin Gly Gly Tyr GIn Gin Tyr Asn Pro Asp Ala Glv Tyr Gin Gin GIn Tyr Asn Prc GIn Gly Gly Tyr Gin Gin Tyr Asn Prco Gin Gl Gly Tyr Gin His Gin Phe Asn Pro Gin Gly Giy Arg Gly Asn Tyr Lys Asn Phe Asn Tyr Asn Asn Asn Leu Gin Gly Tvr Gin Ala Gly WO 2010/079357 PCT/GB2010/050018 63 SEQ ID NO: 55 ggtaIacaaIcc agceagaae~ta C SEQ ID NO: 56 Gly Asn Asn Gin GIn Asn Tyr SEQ ID NO: 57 atgatgatgg cgtctaagga egotacatca agcgtggatg gegetagtgg cgctggtcag ttggtaccgg aggttaatgc ttctgaccet cttgcaatgq atctgtagc aggttcttcg acagcagtcg cqactgtgg acaagtaat cctattgatc cctggataat taataatttt gtgcaagccc cccaaggtga atttactatt tccccaaata atacccccgg tgatgtttg tttgitttgai gtttgggtcc ecacttaat cetttcttgc tocatctatc acaaatgtat aatggttiggg ttggtaacat gagagtcagg attatgetag ctgtaatg cittactgg gggaagataa tagtttcctg cataccccct ggttttggt cacataatct tactatagca caagcaactc tcttccaca tgtgattcgt gatgttagga ctctagaccc cattgaggtg cctttggaag atgttaqgaa tgttctit cataataatg atagaaatca acaaaccatg cgccttgtgt gcatgctgta caccccet: cgcactggtg gtgglactgg tgattctttt gtagttgcag ggcgagttat gacttgcecc: ag9tcctgatt ttatttett gtttttaqtc cctcctacqg tggagcagaa aaccaggcc ttcacactcc caaatotgcc attgagttct ctgtctat cacqtgoccc tctcccaatc algtagtatgg gcatttcccc agacaa tgic cagagtgtgc agttccaaaa tggtcggtgt ac:tc:tggatg gccgcetggt tggcaccacc ccagtttcat tgtcacatqt tgccaaqata agaggacct ccaatggcac tgtaatcaac cttactgaat tggatgqcac accctitcac ccttttgagg gccetgcccc cattgggtUt ccagacctcg gtggtttga ttggcatatc aatatgacac agtttggcea ttctagccag acccagtatg atgtagacac cacccetgac actttigtcc cccatcttqg ttcaattcag gcaaatggca ttggcagtgg taattatgtt ggtgttctta gtggatttc: cec:ccatca caccciuttg gcteccaagt tgacctttgg aagatcecca attatgggtc aagtattacg gaggcaacac atctagcccc tctgtatac ccccctggtt tcggagaggt attggtctt ttcatgtcaa aaatgccagg tcctggtgct t1atttgc cctgtetatt accacaagag tacatttcac atcttgctag tgaacaagcc cctactgtag gtgaggetgc cctgctccac tatgttgacc ctgataccgg tcggaatctt ggggaattca aagcataccc tgatggtttc ctcacttgtg tccccaatgg ggctagctcg ggtcacaac agetgccgat caatggggte ttigtctttg tttcatgggt giccagattt tatcaattaa agcc tgtggg aactqccagc tcggcaagag gitagcttgq tcitggcega taa SEQ ID NO: 58 Met Met Met Ala Ser Lys Asp Ala Thr Ser Ser Val Asp GV Ala Ser Gly Ala Gly Gin Leu Val Pro Glu Val Asn Ala Ser Asp Pro Leu Ala Met Asp Pro Val Ala Gy Ser Ser Thr Ala Val Ala Thr Ala Gly GIn Val Asn Pro Ile Asp Pro Trp Ile lie Asn Asn Phe Val Gin Ala Pro GIn Gv Giu Phe Thr Ile Ser Pro Asn Asn Thr Prc Gly Asp Val Leu Phe Asp Leu Ser Leu Gly Pro His Leu Asn Pro Phe Leu Leu His Leu Ser Gin Met Tyr Asn Gly Trp Val Giv Asn Met Arg Val Arg Ile Met Leu Ala Gly Asn Ala Phe Thr Ala Gly Lys lie Ile Val Ser Cys Ile Pro Pro Gly Phe Gly Ser His Asn Leu Thr lie Ala Gin Ala Thr Leu Phe Pro His Val lie Ala Asp Val Arg Thr Leu Asp Pro Ile Giu Val Pro Leu Glu Asp Val Arg Asn Val Leu Phe His Asn Asn Asp Arg Asn Gin GIn Thr Met Arg Leu Val Cys Met Leu Tyr Thr Pro Leu Arg Thr Gl Giy Gly Thr Gly Asp Ser Phe Val Val Ala Gly Arg Val Met Thr Cys Pro Ser Pro Asp Phe Asn Phe Leu Phe Leu Val Pro Pro Thr Val Gu Gin Lys Thr Arg Pro Phe Thr Leu Pro Asn Leu Pro Leu Ser Ser Leu Ser Asn Ser Arg Ala Pro Leu Pro lie Ser Ser WO 2010/079357 PCT/GB2010/050018 64 Met Gly Ile Ser Pro Asp Asn Val Gin Ser Val Gin Phe Gin Asn Gly Arg Cys Thr Leu Asp Ghy Arg Leu ValGy Thr Thr Pro Val Ser Leu Ser His Val Ala Lys lie Arg Giv Thr Ser Asn Gly Thr Val lie Asn Leu Thr GIU Leu Asp Gly Thr Pro Phe His Pro Phe Glu Gly Pro Ala Pro lie Gly Phe Pro Asp Leu Gly Gly Cys Asp Trp His lie Asn Met Thr GIn Phe GV His Ser Ser GIn Thr GIn Tyr Asp Val Asp Thr Thr Pro Asp Thr Phe Val Pro His Leu Gly Ser lie Gin Ala Asn Gly lIe Gy Ser Gly Asn Tyr Val Gly Val Leu Ser Trp lie Ser Pro Pro Ser His Pro Ser Gly Ser Gin Val Asp Leu Trp Lys lie Pro Asn Tyr Giv Ser Ser lie Thr Giu Ala Thr His Leu Ala Pro Ser Val Tyr Pro Pro Gly Phe Gly Gku Val Leu Val Phe Phe Met Ser Lys Met Pro Gly Pro Gly Ala Tyr Asn Leu Pro Cys Leu Leu Pro GIn Gu Tyr lie Ser His Leu Ala Ser Gu Gin Ala Pro Thr Val Gly Gu AL AL Leu Leu His Tyr Val Asp Pro Asp Thr Gly Arg Asn Leu Gly Glu Phe Lys Ala Tyr Pro Asp Gly Phe Leu Thr Cys Val Pro Asn Gly Ala Ser Ser Gly Pro GIn Gin Leu Pro Ile Asn Gly /al Phe Val Phe Val Ser Trp Val Ser Arg Phe Tyr Gin Leu Lys Pro Val Gly Thr Ala Ser Ser Ala Arg Gly Arg Leu Gl Leu Arg Arg SEQ ID NO: 59 atgatgatgg cttctaaaga Cgctacccz tcttgacg qtgcttctgg tgctqgtcag ctggttccgg aagttaacgc ttctgaccg ctggctatgg acccggttgc tggttcttct accgctgttg ctaccgotgg tcaggttaac ccgatcgacc cgtggatcat caacaac:ttc gttcaggctc cgcagqgtqa attcaccatc tctcegaaca acaCcccggg tgacgttctg ttogacctgt ctctggqtcc gcacctgaac ccgttoctgtgca:c:ctgtc tcacgatglac aacggttggg ttggtaacat gcgtgttcgt atcatgctgg ctggtaacgc tttcaccgct ggtaaaatca tcgtttcttg catcccqccg ggtttceggtt ctcacaacct gaccatoget cagctaccc tgttcccgca cqttatcgct gacgttcgta ccctggaccc gatcgaagtt ccgctggaag acgttcgtaa cgttctgttc cacaacaac-g accgitaacca gcagaccatg cgtctgqttt gcatgctgta caccccgctg ogtaccggtg gtggtaccgg tgactetttc gttgttgctg gtcgtgttat gacctgcccg ttcicggact tcaaettcct gttcctggtt ccgccgaccg ttgaacagaa aacccgtccg ttcaccctgc cgaacctgcc gctgtcttct ctgtetaact otcgtgotcc gctgcogatc tcttctatqg gtatctctcc ggacaacgtt cagtctgtc agttccagaa cggtcgttg accctggacg gtcg tetggt tggtaccacc ccggttictc tgtc Lacgt tgctaaaatc cgtggtacct ctaacggtac cgttatcaac ctgaccgaac tqgacggtac cccgttccac cogttcgaaq gtccggctcc gatcgqttte ccggacctgg gtggttgega ctggcacatc aacatgaccc agtteggtca ctcttotcag acccagtacg acgtigacac caccccggac accttcgtc: cgcacctggg ttctatccag gotaacgga tcggttctgg taactacgtt qgtqttctgt cttggatctc tccgccgtct caccogtctg gttctcaggt tgacctgtgg aaaatcccga actacggttc ttctatcacc: gaagctacc:c acctggctcc gtetg tttac ccgccgggU tcggtgaagt tctggttic ttcatgteta aaatgccqgg tccgggtgct tacaacctgc cgtgcctqt gccgcaqgaa tacatctetc acctggcttc tgaacaggct ecCaccgttg gtgaagctgc tctgctgcac tacgttgacc cggacaccgg tcgtaacctg ggtgaattca aagcttaccc ggacggtttc ctgacetgeg ttccgaacgg tgettottet ggtccgcaqc agccgat caacggtgtt ttcgttttcg ttttgggt ttctcgtttc taccagctga aaccggcttqg taccgcttCt tctgctcgtg gtcgtctggg tctgcgtct tag SEQ ID NO: 60 atgatgatgg ettctaaaga egotacctot tctgttgacg gtgcttctgg igctggtcag ctggttccgg aagttaacgc ttotgacccg etgctatgg acccggttgc tggttcttct accgctgttg ctaccgctgg tcaggttaac ccgatcgacc cgtggatcat caacaacttc gttcaggetc cgcagggtga attcaccate tetecgaaca acaccccgg tqacgttctg ttcgacctgt ctctgggtcc gcacctgaac ccgttcctgc tgcacctgte tcagatgtac aacggttggg ttggtaacat gegtgttcgt atcatgctgg ctggtaacgc tttcacccgct ggtaaaatca tcgittcttg catcccgcg qgtttcggtt ctcacaacct gaccategot caggetaccc tgttcccgca cgttatcqct WO 2010/079357 PCT/GB2010/050018 65 gacgttcgta ccctggaccc gatcgaagtt ccgctggaag acgttcgtaa cgttctgltt cacaacaacg accgtaacca gcagaccatg cqtctgttt gcatgctgta cacccgctg cgtaccggtg gtggtaccgg tgactcttc cttgttgtcgigtcggtat gacctgcccq tctcggact tcaacttcct gttcctggtt cogccgaceg tigaacagaa aacccgtccg ttcaccctgc cgaacctgcc gctgtcttct ctgtctaact etcgtgctcc gctgcegatc tcttctatgg gtatetetcc ggacaacgtt cagtctgttc agttccagaa cggtcgttgc accctggacq gtcgtctggt tggtaccacc ccggtttctc tgtcacgt tgctaaaatc cgtqgtacct ctaacqgtac cgttatcaac ctgaccgaac tggacggtac cccgttccac ccgttcgaag gtccggetcc gatcggtttc ccggacctgg gtggttgcga ctggcacatc aacatgaccc agttcggtca ctctttcag acccagtacg acgtigacac caccccggac accttcgttc cgcacctggg ttctatccag gotaacggta tcggttctgg taactacgtt qgtqttctgt cttggatctc tccgccgtct caccogtctg gttctcaggt tgacctgtgg aaaatcccga actacggttc ttctatcacc: gaagctaccc acctggctcc gtctgtttac ccgccgggtt tcggtgaagt tctggtttic ttcatqtcta aaatgccggg tocgggtgt tacaacctgc cgtqcctct gcgcaqgaa tacatototc acctggcttc tgaacagqt ecgaccgtig gtga:agctgc tctgctgcac tacgttgacc cggacaccgg tcgtaacctg ggtgaattca aagcttaccc ggacggtttc ctgacctqcg ttccgaacqg igcttcttet ggtccgcagco aqgctgccgat caacggtgt ttcgttttcg tttcttgggt ttctogtttc taccagctga aaccggttgg taccgottet tctgctcgtg gtcgtetggq tctgcgtcgt atgatggcct atiggttt tcttggtcca ccgggggcag gcaaaggitac ctatgcgaaa cgittacagg aaatcaccgg catccgcac attaqcacgg gcqacatttt tcqtgatatt gtcaaaaagg aaaatgacga attaggtaag aaaattaaag aaattatgga gogcggcgag ttggtgccgg acgaactggt gaatgaagtt gleaaacgtc ggctgtotga aaaqgattgc gaacgtqggt ttattttqga cggttacccg cgtacaqtag etcaggcaga gttictcgac qggttcctga agactcagaa taaggagtia acggctgcgg tcctgtteoga ggtgcctgaa gaggtggtcg ttcagcgict gaccgcgcgg cgtatctgcc cgaagtqtgg tcgtatttac aacctgattt cacttcctcc aaaagaagat qaactgtgtg atgactgcaa agtaaaactg gtqcaacgcg aagatgataa algaggaaact gtgcegccatc gctacaaagt atatctggaa aaaacccaac cggttatcga ttattatgat aaaaaaggc:a ttttgaaacg cgttgatggg accatcggca tcgataacqt gattqccgaa gttctcaaaa toattqggtg gagtqqataaa SEQ ID NO: 61 Met Met Met Ala Ser Lvs Asp Ala Thr Ser Ser Val Asp Gly Ala Ser Gly Ala Gly Gin Leu Val Pro Glu Val Asn Ala Ser Asp Pro Leu Ala Met Asp Pro Val Ala Gly Ser Ser Thr Ala Val Ala Thr Ala GTy Gin Val Asn Pro lie Asp Pro Trp lie lie Asn Asn Phe Val Gin Ala Pro Gin (y GIu Phe Thr lie Ser Pro Asr Asn Thr Pro Gly Asp Val Leu Phe Asp Leu Ser Leu Giv Pro His Leu Asn Pro Phe Leu Leu His Leu Ser GIn Met Tyr Asn Gly Trp Val Gly Asn Met Arg Val Arg lie Met Leu Ala Gly Asmn Ala Phe Thr Ala Gly Lys lIe Ile Val Ser Cys lie Pro Pro Giy Phe Gly Ser His Asn Leu Thr Ile Ala Gin Ala Thr Leu Phe Pro His Val le Ala Asp Val Arg Thr Leu Asp Pro lie Giu Val Pro Leu Glu Asp Val Arg Asn Val Leu Phe His Asn Asn Asp Arg Asn GIn GIn Thr Met Arg Leu Val Cys Met Leu Tyr Thr Pro Leu Arg Thr -Gly Gly Gly Thr Gly Asp Ser Phe Val Val Ala Gly Arg Val Met Thr Cys Pro Ser Pro Asp Phe Asn Phe Leu Phe Leu Val Pro Pro Thr Val Gu Gin Lys Thr Arg Pro Phe Thr Leu Pro Asn Leu Pro Leu Ser Ser Leu Ser Asn Ser Arg Ala Prc Leu Pro Ile Ser Ser Met Glv Ie Ser Pro Asp Asn Val Gin Ser Val Gin Phe Gin Asn Gly Arg Cys Thr Leu Asp Gly Arg Leu Val Gly Thr Thr Pro Val Ser Leu Ser His Val Ala Lys Ile Arg Gly Thr Ser Asn Gly Thr Val lie Asn Leu Thr Glu Leu Asp Gly Thr Pro Phe His Pro Phe Giu Gly Pro Ala Pro ile Gly Phe Pro Asp Leu Gly Gly Cys Asp Trp His Ile Asn Met Thr Gin Phe Gly His Ser Ser Gin Thr Gin Tyr Asp Val Asp Thr Thr Pro Asp Thr Phe Val Pro His Leu Gly Ser lie Gin Ala Asn Gly Ile Gly Ser Gly Asn Tyr Val Gly Val Leu Ser Trp Ile Ser Pro Pro Ser His Pro Ser GlV Ser GIn Val Asp Leu Trp Lys lIe Pro Asn Tyr GlV Ser Ser lIe Thr Giu Ala Thr His Leu WO 2010/079357 PCT/GB2010/050018 66 Ala Pro Ser Val Tyr Pro Pro Gly Phe Gly Glu Val Leu Val Phe Phe Met Ser Lys Met Pro Gly Pro Gly Ala Tyr Asn Leu Pro Cys Leu Leu Pro Giln Gilu Tyr lie Ser His Leu Ala Ser Gilu GIn Ala Pro Thr Val Gly Glu Ala Ala Leu Leu His Tyr Val Asp Pro Asp Thr Gly Arg Asn Leu Gly Glu Phe Lys Ala Tyr Pro Asp Glv Phe Leu Thr Cys Val Pro Asn Gly Ala Ser Ser Gly Pro Gin GIn Leu Pro lie Asn Gly Val Phe Val Phe Val Ser Trp Val Ser Arq Phe Tyr Gin Leu Lys Pro Val Gly Thr Ala Ser Ser Ala Arg Gly Arg Leu Gly Leu Arg Arg Met Met Ala Tyr Leu Val Phe Leu Gly Pro Pro Gy Ala Gli Lvs Gly Thr Tyr Ala Lys Arg Leu Gin Glu lie Thr Giy lie Pro His lie Ser Thr Gly Asp lie Phe Arg Asp le Val Lys Lys Giu Asn Asp G3u Leu Gly Lys Lys lie Lys Glu lie Met Glu Arg Gly Gu Leu Val Pro Asp Glu Leu Val Asn Glu Val Val Lys Arg Arg Leu Ser Gu Lys Asp Cys Gu Arg Gly Phe lie Leu Asp Gly Tyr Pro Arg Thr Val Ala Gin Ala Gu Phe Leu Asp Gly Phe Leu Lys Thr Gin Asn Lys Gu Leu Thr Ala Ala Val Leu Phe Giu Val Pro Gu Gliu Val Val Val Gin Arg Leu Thr Ala Arg Arg lie Cys Pro Lys Cys Gly Arg lie Tyr Asn Leu lie Ser Leu Pro Pro Lys Glu Asp Gki Leu Cys Asp Asp Cys Lys Val Lys Leu Val Gin Arg Glu Asp Asp Lys Gu Giu Thr /al Arg His Arg Tyr Lys Val Tyr Leu Glu Lys Thr Gin Pro Val lIe Asp Tyr Tyr Asp Lys Lys Gly lIe Leu Lys Arg Val Asp Gly Thr lie Gly IE Asp Asn Val lie Ala Giu Val Leu Lys Ile Ile GIy Tip Ser Asp Lys SEQ ID NO: 62 Met Ala Ser Asn Phe Thr Gin Phe Val Leu Val Asp Asn Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Giu Trp lie Ser Ser Asn Ser Arg Ser Gin Ala Tyr Lys Val Thr Cys Ser /al Arq Gin Ser Ser Ala GIn Asn Arg Lys Tyr Thr Ile Lys Val Giu Val Pro Lys Val Ala Thr Gin Thr Val Gly GIy Val Gu Leu Pro Val Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr lie Pro lie Phe Ala Thr Asn Ser Asp Cys Giu Leu Ile Val Lys Ala Met Giln Gly Leu Leu Lys Asp Gly Asn Prc lie Pro Ser Ala lie Ala Ala Asn Ser Glv Ile Tvr SEQ ID NO: 63 Ser Lys Thr Ile Val Leu Ser Val GV Glu Ala Thr Arg Thr Leu Thr Giu lie Gin Ser Thr Ala Asp Arg Gin lie Phe Glu Gu Lys Val Gly Pro Leu Val Gly Arg Leu Arq Leu Thr Ala Ser Leu Arg Gin Asn Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gin Ala Asp Val Val Asp Cys Ser Thr Ser Val Cys Gly Giu Leu Pro Lys Val Arg Tyr Thr Gin Val Trp Ser His Asp Val Thr lIe Val Ala Asn Ser Thr Giu Ala Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val Val Gin Ala Thr Ser Glu Asp Leu Val Val Asn Leu Val Pro Leu Gly Arg SEQ ID NO: 64 Met Ser Lys Thr lie Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu Thr Glu lIe Gin Ser Thr Ala Asp Arg Gin lie Phe Glu Glu Lys Val Glv Pro Leu Val Gly Arm Leu Arg Leu Thr Ala Ser Leu Arg Gin Asn Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gin Ala Asp Val Val Asp Ser Gy Leu Pro Lys Val Arg Tyr Thr GIn Val Trp Ser His Asp Val Thr Ile Val Ala Asn Ser Thr Glu Ala Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val Ala Thr Ser Gin Val Giu Asp Leu Val Val Asn Leu Val Pro Leu( Gly Arg Tyr Gly Ser Lys Thr lie Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu Thr Giu Ile Gin Ser Thr Ala Asp Arg Gin Ile Phe Gu Glu Lys Val Gly Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gin Asn Gly Ala Lys Thr Ala Tyr Arg Val WO 2010/079357 PCT/GB2010/050018 67 Asn Leu Lys Leu Asp Giri Ala Asp Val Val Asp Ser Ghy Leu Pro Lys Val Arg Tyr Thr Gin Val Trp Ser His Asp Val Thr lie Val Ala Asn Ser Thr Glu Ala Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val Ala Thr Ser Gin Val Giu Asp Leu Val Val Asn Leu Val Pro Leu Gly Arg SEQ ID NO: 65 Met Lys Leu Len Lys Val Ala Ala lie Ala Ala lie Val Phe Ser Gly Ser Ala Leu Ala Gly Val Val Pro Gin Tyr Gly Gly Gly Gly Asn His Gly Gly Gly Gly Asn Asn Ser Gly Pro Asn Ser Giu Leu Asn lie T yr Gin T y'r -Gy Gly Gly Asn Ser Ala Leu Ala Leu Gin Thr Asp Ala Arg Asn Ser Asp Leu Thr lie Thr Gin His Gly Gly Gly Asn Giv Ala Asp Val Gly Gin Gly Ser Asp Asp Ser Ser lie Asp Leu Thr Gin Arg Gly Phe Gly Asn Ser Ala Thr Leu Asp Gin Trp Asn Gly Lys Asn Ser Giu Met Thr Val Lys Gin Phe Gly Gly Gly Asn Gly Ala Ala Val Asp GIn Thr Ala Ser Asn Ser Ser Val Asn Val Thr Gin Val Gly Phe Gly Asn Asn Ala Thr Ala His Gin Tyr SEQ ID NO: 66 Met Lys Leu Leu Lys Val Ala Ala Phe Ala Ala lie Val Val Ser Gly Ser Ala Leu Ala Gly Val Val Pro Gin Trp Gly Gly Gly Gly Asn His Asn Gly Gly Gly Asn Ser Ser Gly Pro Asp Ser Thr Leu Ser lie Tyr Gin Tyr Gly Ser Ala Asn Ala Ala Leu Ala Leu Gin 3cr Asp Ala Arg Lys Ser Giu Thr Thr lie Thr Gin Ser Gly Tyr Gly Asn Gly Ala Asp Val Gly Gin Gly Ala Asp Asn Ser Thr lIe Gin Leu Thr Gin Asn Gly Phe Arg Asn Asn Ala Thr lie Asp GIn Trp Asn Ala Lys Asn Ser Asp lIe Thr Val Gly Gin Tyr Gly Gly Asn Asn Ala Ala Leu Val Asn GIn Thr Ala Ser Asp Ser Ser Val Met Val Arg GIn Val Gly Phe Gly Asn Asn Ala Thr Ala Asn Gin Tvr SEQ ID NO: 67 Met Met Ala Tyr Leu Val Phe Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Tyr Ala Lys Arg lie Gin Giu Lys Thr Gly lie Pro His lIe Ser Thr Gly Asp lie Phe Arg Asp lie /al Lys Lys Giu Asn Asp GIu Leu Gly Lys Lys lIe Lys Giu lie Met GIu Lys Gly Giu Leu /al Pro Asp Gin Leu Val Asn Giu Val Val Lys Arg Arg Leu Ser GIu Lys Asp Cys Giu Lys Gly Phe lie Leu Asp Gly Tyr Pro Arq Thr Val Ala Gin Ala GIu Phe Leu Asp Ser Phe Leu Giu Ser Gin Asn Lys Gin Leu Thr Ala Ala Val Leu Phe Asp Val Pro Glu Asp Val Val Val Gin Arg Leu Thr Ser Arg Arg lIe Cys Pro Lys Cys GIy Arg lie Tyr Asn Met lie Ser Leu Pro Pro Lys Giu Asp Giu Leu Cys Asp Asp Cys Lys 'al Lys Leu Val Gin Arm Asp Asp Asp Lys Giu Giu Thr Val Arg His Arg Tyr Lys Val Tyr Leu Glu Lys Thr GIn Pro Val lie Asp Tyr Tyr Gly Lys Lys Gly lie Leu Lys Arg Val Asp Gly Thr lie Gly lie Asp Asn Val Val Ala Giu Val Leu Lys lie lie Gly Trp Ser Asp Lys Gly Ser GIN Val Val Pro Gin Tyr Gly Gly Gly Gly Asn His Gly Giy Gly Gly Asn Asn Ser GIy Pro Asn Ser Giu Leu Asr lie Tyr Gin Tyr Giy Gly Gly Asn Ser Ala Leu Ala Leu Gin Thr Asp Ala Arg Asn Ser Asp Leu Thr iie Thr Gin His Gly Gly Gly Asn Gly Ala Asp Val Gly Gin Gly Ser Asp Asp Ser Ser lie Asp Leu Thr Gin Arg Gly Phe Gly Asn Ser Ala Thr Len Asp Gin Trp Asn Gly Lys Asn Ser Giu Met Thr Val Lys Gin Phe Gly Gly Gly Asn Gly Ala Ala Val Asp Gin Thr Ala Ser Asn Ser Ser Val Asn Val Thr GIn Val Gly Phe Gly Asn Asn Ala Thr Ala His Gin Tyr SEQ ID NO: 68 WO 2010/079357 PCT/GB2010/050018 68 Met Gin Phe Ser Thr Leu Thr Thr Val Phe Ala Leu Val Ala Ala Ala Val Ala Ala Pro His Gly Ser Ser Gly Gly Asn Asn Pro Val Cys Ser Ala Gin Asn Asn Gin Val Cys Cys Asn GlV Leu Leu Ser Cys Ala Val Gin Val Leu Gly Ser Asn Cys Asn Gly Asn Ala Tyr Cys Cys Asn Thr Glu Ala Pro Thr Gly Thr Leu ile Asn Val Ala Leu Leu Asn Cys Val Lys Leu Leu SEQ ID NO: 69 Met Lys Phe Ser Leu Ala Ala Val Ala Leu Leu Sly Ala Val Val Ser Ala Leu Pro AL Asn Giu Lys Arg Gin Ala Tyr lie Pro Cs Ser GIy Leu Tyr Gly Thr Ser Gin Cys Cys Ala Thr Asp Val Leu Gly Val Ala Asp Leu Asp Cys Giy Asn Pro Pro Ser Ser Pro Thr Asp Ala Asp Asn Phe Ser Ala Val Cys Ala Glu lie GBy Gin Arg Ala Arg Cys Cys Val Leu Pro ie Leu Asp Gin Gly ie Leu Cys Asn Thr Pro Thr Gly Val Gin Asp SEQ ID NO: 70 Val Pro Pro Pro Cys Asp Leu Ser lIe Lys Ser Lys Leu Lys Gin Val Gly Ala Thr Ala Gly Asn Ala Ala Val Thr Thr Thr Gly Thr Thr Ser Gly Ser Gy Val Val Lys Cys Val Val Arg Thr Pro Thr Ser Val Giu Lys Lys Ala Ala Val Giy Asn Thr Giy Leu Ser Ala Val Ser Ala Ser Ala Ala Asn Gy Phe Phe Lys Asn Leu Gl, Lys Ala Thr Thr Glu Val Lys Thr Thr Lys Asp Gly Thr Lys Val Lys Thr Lys Thr Ala Gy Lys Gly Lys Thr Gly Gy Thr Ala Thr Thr lie GIn Ile Ala Asp Ala Asn Gly Gly Val Ser Glu Lys Ser Leu Lys Leu Asp Leu Leu Thr Asp Sly Leu Lys Phe Val Lys Val Thr Glu Lys Lys Gin Giv Thr Ala Thr Ser Ser Ser Gly His Lys Ala Ser Gy Val Gly His Ser Val Phe Lys Val Leu Asn Glu Ala Glu Thr Giu Leu Glu Leu Lys Gly Leu SEQ ID NO: 71 Met Lys Trp Phe Leu Phe Leu Leu Thr Thr Ala Val Leu Ala Ala /aI /al Ser Ala His Giu Glu Asp Gly Val Cys Asn Ser Asn AL Pro Cys Tyr i-s C ys Asp Ala Asn Gly Slu Asn Cys Ser Cys Asn Cys Glu Leu Phe Asp Cys Glu Ala Lys Lys Pro Asp Gly Ser Tyr Ala His Pro Cys Arg Arg Cys Asp Ala Asn Asn lie Cys Lys Cys Ser Cys Thr Ala lie Pro Cys Asn Glu Asp His Pro Cys His His Cys HisGlu Glu Asp Asp Gly Asp Thr His Cys His Cys Ser Cys Gu His Ser His Asp His His Asp Asp Asp Thr His Gly Gu Cys Thr Lys Lys Ala Pro Cys Trp Arg Cys Glu Tyr Asn Ala Asp Leu Lys His Asp Val Cys Gly Cys Gu Cys Ser Lys Leu Pro Cys Asn Asp Giu His Pro Cys Tyr Arg Lys Glu Gly Gly Val Val Ser Cys Asp Cys Lys Thr lie Thr Cys Asn Giu Asp His Pro Cys Tyr His Ser Tyr Gu Glu Asp Gly Val Thr Lys Ser Asp Cys Asp Cys Glu His Ser Pro Gly Pro Ser Gu SEQ ID NO: 72 Met Arg Val Leu Val lie Asn Ser Giv Ser Ser Ser lie Lys Tyr Gin Leu lie Giu Met Glu Gly Glu Lys Val Leu Cys Lys Gly lie Ala Glu Arq le Gly lie Giu Gly Ser Arg Leu Val His Arg Val Gly Asp Giu Lys His Val lIe Giu Arg: Gu Leu Pro Asp His Glu Gu Ala Leu Lys Leu lie Leu Asn Thr Leu Val Asp Glu Lys Leu Gly Val Ile Lys Asp Leu Lys Glu lie Asp Ala Val Gly His Arg Val Val His Gly Gly Glu Arg Phe Lys GIu Ser Val Leu Val Asp Gu Gu Val Leu Lys Ala lie Gu Giu Val Ser Pro Leu Ala Pro Leu His Asn Pro Ala Asn Leu Met Cly lie WO 2010/079357 PCT/GB2010/050018 69 Lys Ala Ala Met Lys Leu Leu Pro Gly Val Pro Asn Val Ala Val Phe Asp Thr Ala Phe His Gin Thr lie Pro Gin Lys Ala Tyr Leu Tyr Ala lie Pro Tyr Giu Tyr Tyr Glu Lys Tyr Lys lie Arg Arg Tyr Gly Phe His Gly Thr Ser His Arg Tyr Val Ser Lys Arg Ala Ala Gu lie Leu Gly Lys Lys Leu Giu Giu Leu Lys lie lie Thr Cys His lie Gy Asn Gly Ala Ser Val Ala Ala Vail Lys Tyr Gly Lys Cys Val Asp Thr Ser Met GlV Phe Thr Pro Leu Glu (y Leu Val Met Gly Thr Arg Ser Gly Asp Leu Asp Pro Ala lie Pro Phe Phe lie Met Gu Lys GIu Giy lie Ser Pro Gin Glu Met Tyr Asp lie Leu Asn Lys Lys Ser Gly Val Tyr Gly Leu Ser Lys Gv Phe Ser Ser Asp Met Arg Asp lie G1u Giu Ala Ala Leu Lys Glv Asp Glu Trp Cys Lys Leu Val Leu GIu lie Tyr Asp Tyr Arg lie Ala Lys Tyr lie Gly Ala Tyr Ala Ala Ala Met Asn Gly Val Asp Ala Ile Val Phe Thr Ala Gly Val Gly Glu Asn Ser Pro Ile Thr Arg GIu Asp Val Cys Ser Tyr Leu GIu Phe Leu Glv Val Lys Leu Asp Lvs GIn Lys Asn Giu Giu Thr lie Arg Gly Lys Giu Gly lIe lie Ser Thr Pro Asp Ser Arg Val Lys Val Leu Val Val Pro Thr Asn Glu Giu Leu Met lie Ala Arg Asp Thr Lys Glu lie Val Giu Lys lIe Gly Arg Val Pro Pro Pro Cys Asp Leu Ser lie Lys Ser Lys Leu Lys Gin /al Gly Ala Thr Ala Gly Asn Ala Ala Val Thr Thr Thr Gly Thr Thr Ser Gly Ser Gly VaI I Lys Cys Val Val Arg Thr Pro Thr Ser Val iu Lys Lys Ala Ala Val Gly Asn Thr Gly Leu Ser Ala Val Ser Ala Ser Ala Ala Asn Glv Phe Phe Lys Asn Leu Gly Lys Ala Thr Thr Glu Val Lys Thr Thr Lys Asp Gly Thr Lys Val Lys Thr Lys Thr Ala Gly Lys Gly Lys Thr Gly Gly T hr Ala Thr Thr Ile Gin Ile Ala Asp AL Asn Glv Gy Val Ser Gu Lys Leu Lys Leu Asp Leu Leu Thr Asp Gly Leu Lys Phe Val Lys Val Thr Glu Lys Lys Gin Gly Thr Ala Thr Ser Ser Ser Gly His Lys Ala Ser Gly Val Gly His Ser Val Phe Lys Val Leu Asn Giu Ala Gliu Thr Giu Leu Gu Leu Lys Glv Leu SEQ ID NO: 73 Val Pro Pro Pro Cys Asp Leu Ser lie Lys Ser Lys Leu Lys Gin Val Gly Ala Thr Ala Gly Asn Ala Ala Val Thr Thr Thr Glv Thr Thr Ser Gly Ser Gly Val Val Lys Cys Val Val Arg Thr Pro Thr Ser Val Gu Lys Lys Ala Ala Val Gly Asn Thr Gly Leu Ser Ala Val Ser Ala Ser Ala Ala Asn Gly Phe Phe Lys Asn Leu Gly Lys Ala Thr Thr Glu Val Lys Thr Thr Lys Asp Gly Thr Lys Val Lys Thr Lys Thr Ala Gly Lys Gly Lys Thr Gly Gly Thr Ala Thr Thr lie Gin lie Ala Asp Ala Asn Gly Gly Val Ser GIu Lys Ser Leu Lys Leu Asp Leu Leu Thr Asp Gly Leu Lys Phe Val Lys /al Thr Glu Lys Lys GIn GlV Thr Ala Thr Ser Ser Ser Gly His Lys Ala Ser Gly Val Gly His Ser Val Phe Lys Val Leu Glu Ala GIu Thr -GIu Leu Glu Leu Lys Gly Leu Met Arg Val Leu Va lie Asn Ser Gly Ser Ser Ser lie Lys Tyr Gin Leu Ile Glu Met GILu Gly GIu Lys Val Leu Cys Lys Gly ile Ala GIu Arg lie Gly lie Glu Gy Ser Arg Leu Val His Arg Val Gly Asp Gu Lys His Val lie Gilu Arg Gu Leu Pro Asp His Glu Giu Ala Leu Lys Leu lle Leu Asn Thr Leu Val Asp Glu Lys Leu Gly Val lie Lys Asp Leu Lys GIu lie Asp Ala Val Gly His Arg Val Val His Gly Gly Gu Arg Phe Lys Gu Ser Val Leu Val AspGlu G u Val Leu Lys Ala lie GIU GLu Val Ser Pro Leu Ala Pro Leu His Asn Pro Ala Asn Leu Met Gy lie Lys Ala Ala Met Lys Leu Leu Pro Gly Val Pro Asn Val Ala Val Phe Asp Thr Ala Phe His Gin Thr lie Pro Gin Lys Ala Tyr Leu Tyr Ala Ile Pro Tyr Giu Tyr Tyr Glu Lys Tyr Lys lie Arg Arg Tyr Gly Phe His Gly Thr Ser His Arg Tyr Val Ser Lys Arg Ala Ala Giu lie Leu Giv Lys Lvs Leu Gu Glu Leu Lys lie Ile Thr Cys His lie Gly Asn Gly Ala Ser Val Ala Ala Val Lys Tyr Gly Lys Cys Val Asp Thr Ser Met Gly Phe Thr Pro Leu GIU Gly Leu Val Met Gly Thr Arg Ser Gly Asp Leu Asp Pro Ala lie Pro Phe Phe lie Met Giu Lys Glu Gly lie Ser Pro Gin Glu Met Tyr Asp Ile Leu Asn Lys Lys Ser Gly Val Tyr Gly Leu Ser Lys Gly Phe Ser SEr Asp Met Arg Asp lie Giu GIu Ala Ala Leu WO 2010/079357 PCT/GB2010/050018 70 Lys Gly Asp Glu Trp Cys Lys Leu Val Leu Giu lie Tyr Asp Tyr Arg Ile Ala Lys Tyr le Gly Ala Tyr Ala Ala Ala Met Asn Glv Val Asp Ala lie Val Phe Thr Ala Gly Val Gly Glu Asn Ser Pro lie Thr Arg Glu Asp Val Cys Ser Tyr Leu GIu Phe Leu Gly Val Lys Leu Asp Lys Gin Lys Asn Giu Giu Thr lie Arg Gly Lys Gliu Gly lie lie Ser Thr Pro Asp Ser Arg Val Lys Val Leu Val Val Pro Thr Asn Gu Gu Leu Met lie Ala Arg Asp Thr Lys GIu lie Val Glu Lys lie Gly Arg SEQ ID NO: 74 Met Lys Tyr Thr Leu Ala Leu Leu Phe Leu Thr Ala lie Ile Ala Thr Phe Val Ala Ala His Lys His His Asp His Giy Lys Ser Cys Ser Lys Ser His Pro Cys Tyr His Cys His Thr Asp Cys Gu Cys Asn His His His Asp .Asp Cys Asn Arg Ser His Arg Cys Trp His Lys Val His (Thy Val Val Ser Gly Asn Cys Asn Cys Asn Leu Leu Thr Pro Cys Asn Gin Lys His Pro Cys Trp Arg Arg His Gly Lys Lys His Gly Leu His Arg Lys Phe His Gby Asn Ala Cys Asn Cys Asp Arg Leu Val Cys Asn Ala Lys His Pro Cys Trp His Lys His Cys Asp Cys Phe Cys SEQ ID NO: 75 Ser Lys Leu Pro Cys Asn Asp Gu His Pro Cys Fr Arg Lys Giu Gly Gly Val Val Ser Cys Asp Cys Lys SEQ ID NO: 76 Ser Lys Leu Pro Ser Asn Asp Giu His Pro Ser Tyr Arg Lys Glu lv Glv Va al Ser Ser Asp Ser Lys SEQ ID NO: 77 Lys Thr Ile Thr Cys Asn Gu Asp His Pro Cys Tyr His Ser Tyr Giu GIu Asp Gly Val Thr Lys Ser Asp Cys Asp Cvs Glu SEQ ID NO: 78 Met Arg lie lie Leu Leu Gly Ala Pro Gly Ala Gly Lys Gly Thr Gin AL Gn Phe lie Met Giu Lys Tyr Gly lie Pro Gin lie Ser Thr Gly Asp Met Leu Arg Ala Ala Val Lys Ser GT;y Ser Giu Leu Gly Lys Gin Ala Lys Asp lie Met Asp Ala Gly Lys Leu Val Thr Asp Gu Leu Val Ile Ala Leu Val Lys Giu Arg lie Ala Gin Giu Asp Cys Arg Asn Gly Phe Leu Leu Asp Gly Phe Pro Arg Thr lie Pro Gin Ala Asp Ala Met Lys Gu Ala Gy lie Asn Val Asp Tyr Val Leu Glu Phe Asp Val Pro Asp Glu Leu lie Val Asp Arg Ile Val Gly Arg Arg Val His Ala Pro Ser Gly Arg Val Tyr His Val Lys Phe Asn Pro Pro Lys Val Glu Gly Lys Asp Asp Val Thr Gly Gliu Gu Leu Thr Thr Arg Lys Asp Asp GIn Glu Gilu Thr Val Arg Lys Arg Leu Val Gu Tyr His Gin Met Thr Ala Pro Leu lie Gy Tyr Tyr Ser Lys Glu Ala Giu Ala Gly Asn Thr Lys Tyr Ala Lys Val Asp Gly Thr Lys Pro Val Ala Giu Val Arg Ala Asp Leu Glu Lys lie Leu GlV SEQ ID NO: 79 WO 2010/079357 PCT/GB2010/050018 71 Met Lys Lys Thr Lys lie Val Cys Thr lie Gly Pro Lys Thr Giu Ser Glu GIu Met Leu Ala Lys Met Leu Asp Ala Gly Met Asn k/al Met Arg Leu Asn Phe Ser His Gly Asp Tyr Ala Giu His Glv Gin Arg lie Gin Asn Leu Arg Asn Val Met Ser Lys Thr Gly Lys Thr Ala Ala Ile Leu Leu Asp Thr Lys Gly Pro Glu lie Arg Thr Met Lys Leu Glu Glv GLy Asn Asp k/al Ser Leu Lys Ala Gly GIn Thr Phe Thr Phe Thr Thr Asp Lys Ser Val lie Glv Asn Ser Glu Met V/al Ala Val Thr Tyr Glu Gly Phe Thr Thr Asp Leu Ser Val Giy Asr Thr Val Leu Val Asp Asp Glv Leu lie Gly Met Glu Val Thr Ala lie Glu Gly Asn Lys /al lie Cvs Lys Val Leu Asn Asn Giy Asp Leu Gly Glu Asn Lys Gly Val Asn Leu Pro Gly Val Ser lie Ala Leu Pro Ala Leu Ala Giu Lys Asp Lys Gin Asp Leu lIe Phe Gly Cys Glu Gin Gly Val Asp Phe Val Ala Ala Ser Phe lie Arg Lys Arg Ser Asp Va lie Giu lIe Arg Glu His Leu Lys Ala His Gly Gly Gu Asn lIe His lie lIe Ser Lys lie Glu Asn Gin Giu -Gly Leu Asn Asn Phe Asp Giu lIe Leu Gu Ala Ser Asp Gly lIe Met Val Ala Arg Gly Asp Leu Gy Val GIu lie Pro Val Gliu Gu Val lie Phe Ala Gin Lys Met Met lie Gu Lys Cys lie Arg Ala Arg Lys Val Val lie *Thr Ala Thr Gin Met Leu Asp Ser Met lie Lys Asn Pro Arq Pro Thr Arg Ala Giu Ala Gly Asp /al Ala Asn Ala lie Leu Asp Gly Thr Asp Ala Val Met Leu Ser Giy GIu SEr Ala Lys Gly Lys Tyr Pro Leu Giu Ala V/al Ser ile Met Ala Thr ie Cys Giu Arg Thr Asp Arg Val Met Asn Ser Arg Leu Gu Phe Asn Asn Asp Asn Arg Lys Leu Arg lie Thr Gu Ala Val Cys Arg Giy Ala Val G1u Thr Ala Glu Lys Leu Asp Ala Pro Leu lie Val Val Ala Thr GIn Giv Gly Lys Ser Ala Arm Ala /al Arm Lys Tyr Phe Pro Asp Ala Thr lie Leu Ala Leu Thr Thr Asn Glu Lys Thr Ala His Gin Leu Val Leu Ser Lvs Glv Val Val Pro Gin Leu Val Lvs Glu lie Thr Ser Thr Asp Asp Phe Tyr Arg Leu Gly Lys Gu Leu Ala Leu Gin Ser Gly Leu Ala His Lys Gly Asp Val Val /al Met Val Ser Gly Ala Leu Val Pro Ser Gly Thr Thr Asn Thr Ala Ser Val His Val Leu SEQ ID NO: 80 Met Ser Ser Lys Leu /al Leu Val Leu Asn Cys Giv Ser Ser Ser Leu Lvs Phe Ala lIe lie Asp Ala Val Asn Gly Gu Giu Tyr Leu Ser Gly Leu Ala Giu Cys Phe is Leu Pro Glu Ala Arg Ile Lys Trp Lys Met Asp Gly Asn Lys Gin Giu Ala Ala Leu GIV Ala Gly Ala Ala His Ser Giu Ala Leu Asn Phe lie /al Asn Thr lie Leu Ala Gin Lys Pro Gu Leu Ser Ala Gin Leu Thr Ala lIe Gly His Arg lie Val His Gy Gly Glu Lys Tyr Thr Ser Ser Val Val lIe Asp Gliu Ser Val lie GIn Gly lIe Lys Asp Ala Ala Ser Phe Ala Pro Leu His Asn Pro Ala His Leu lie Glv lie Glu Gu Ala Leu Lys Ser Phe Pro Gn Leu Lys Asp Lys Asn Val Ala Val Phe Asp Thr Ala Phe His Gin Thr Met Pro GIu GIu Ser Tyr Leu Tyr Ala Leu Pro Tyr Asn Leu Tyr Lys Giu His Gly lie Arg Arg Tyr Giv Ala His Giv Thr Ser His Phe Tyr Val Thr Gin Gu Ala Ala Lys Met Leu Asn Lys Pro Val Gu Glu Leu Asn le Ile Thr Cys His Leu Gly Asn Gly Gly Ser Val Ser Ala lie Arg Asn Gly Lys Cys Val Asp Thr Ser Met Gly Leu Thr Prc Leu Glu Gly Leu Val Met Gy Thr Arg Ser Gly Asp lie Asp Pro Ala lie Ile Phe His Leu His Asp Thr Leu Gly Met Ser Val Asp Ala lie Asn Lys Leu Leu Thr Lys Giu Ser Gly Leu Leu Gly Leu Thr Glu Val Thr Ser Asp Cys Arg Tyr Val Gu Asp Asn Tyr Ala Thr Lys Giu Asp Ala Lvs Arg Ala Met Asp Val Tyr Cys His Arg Leu Ala Lys Tyr lie Gly Ala Tyr Thr Ala Leu Met Asp Gly Arg Leu Asp Ala Val Val Phe Thr Gly Gly lie Gly Gu Asn Ala Aia Met Val Arg TGu Leu Ser Leu Gly Lys Leu Gly Val Leu Gly Phe Glu Val Asp His Giu Arg Asn Leu Ala Ala Arg Phe Gly Lys Ser Gly Phe lie Asr Lys Giu Gly Thr Arq Pro AL Val Val lIe Pro Thr Asn Gu Glu Leu Val lie Ala Gin Asp Aia Ser Arg Leu Thr Ala WO 2010/079357 PCT/GB2010/050018 72 SEQ ID NO: 81 Met Lys Asn Lys Val Val Val Val Thr Gly Val Pro Gly Val Gly Ser Thr Thr Ser Ser Gin Leu Ala Met Asp Asn Leu Arg Lys Giu Giv Val Asmn Tyr Lys Met /al Ser Phe Gly Ser /al Met Phe Giu Val Ala Lys Giu Giu Asn Leu Val Ser Asp Arg Asp Gin Met Arg Lys Met Asp Pro Gu Thr Gin Lys Arg Ile Gin Lys Met Ala Gly Arm Lys Ile Ala Glu Met Ala Lys Glu Ser Pro Val Ala Val Asp Thr His Ser Thr Val Ser Thr Pro Lys Gly Tyr Leu Pro Giy Leu Pro Ser Trp Val Leu Asn Glu Leu Asr Pro Asp Leu lie lie Val Vat Glu Thr Thr Gly Asp Giu lie Leu Met Arg Arg Met Ser Asp Giu Thr Arg Vail Arg Asp Leu Asp Thr Ala Ser Thr lie Giu Gn His GIn Phe Met Asn Arg Cys Ala Ala Met Set Tyr Gly Val Leu Thr Gly Ala Thr Val Lys lHe Val Gin Asn Arg Asn Gly Leu Leu Asp GIn Ala Val Gu Gu Leu Thr Asn Val Leu Arg SEQ ID NO: 82 Met Lys Asn Lys Leu Val Val /al Thr Gly Val Pro Gly Val Gly Glv Thr Thr le 1hr Gin Lys Ala Met Giu Lys Leu Ser Gu Giu Gly lie Asn Tyr Lys Met Val Asn Phe Gly Thr /al Met Phe Gu Val Ala Gin Giu Giu Asn Leu Val Gu Asp Arg Asp GIn Met Arg Lys Leu Asp Pro Asp Thr Gin Lys Arg Ile Gin Lys Leu Ala Gly Arg L ys lie Ala Giu MetVal Lys Gu Ser Pro Val Val Val Asp Thr His Ser Thr lie Lys Thr Pro Lys Gly Tyr Leu Pro Gly Leu Pro Val Trp Val Leu Asn Giu Leu Asn Pro Asp Ile lIe Ile Val Val Giu Thr Ser GIy Asp Giu lie Leu lie Arg Arg Leu Asn Asp Giu Thr Arm Asn Arq Asp Leu Gu Thr Thr Ala Gly lie Giu Giu His Gin lie Met Asn Arg Ala Ala Ala Met Thr Tyr Gy Val Leu Thr GlV Ala Thr Val Lys lIe Ile Gin Asn Lys Asn Asn Leu Leu Asp Tyr Ala Val Gu Glu Leu Ile Ser Val Leu Arg SEQ ID NO: 83 Met Asn lie Val Leu Met Giy Leu Pro GlV Ala Gv Lys Gly Thr Gin Ala Asp Arg Ile Val Glu Lys Tyr Gly Thr Pro His Ile Ser Thr Gly Asp Met Phe Arg Ala Ala lie Gin Glu Gly Thr Glu Leu Gly Val Lys Ala Lys Ser Phe Met Asp GIn Gly Ala Leu Val Pro Asp Gu Val Thr lIe Gly lie Val Arg Ghu Arg Leu Ser Lys Ser Asp Cys Asp Asn Gly Phe Leu Leu Asp Gly Phe Pro Arg Thr Val Pro Gin Ala Giu Ala Leu Asp Gin Leu Leu Ala Asp Met Gly Arg Lys lie Gu His Val Leu Asn lIe Gin Val Gu Lys Gh Giu Leu lie Ala Arg Leu Thr Gly Arg Arg lie Cys Lys Val Cys Gly Thr Ser Tyr His Leu Leu Phe Asn Pro Pro Gin Val Giu Gly Lys Cvs Asp Lys Asp Gly Gly Glu Leu Tyr Gin Arg Ala Asp Asp Asri Pro Asp Thr Val Thr Asn Arg Leu Giu Val Asn Met Asn Gin Thr Ala Pro Leu Leu Ala Phe Tyr Asp Ser Lys Glu Val Leu Val Asn le Asn Gly Gin Lys Asp Ile Lys Asp Val Phe Lys Asp Leu Asp Val lie Leu Gin Gly Asn GlV Gin SEQ ID NO: 84 Met Asn Leu /al Leu Met Gly Leu Pro (;y Ala (y Lys Gly Thr GIn Gly Gu Arg lie Val Gliu Asp Tyr Gly lie Pro His lie Ser Thr Glv Asp Met Phe Arg Ala Ala Met Lys Glu Glu Thr Pro Leu Gly Leu Gu Ala Lys Ser Tyr lie Asp Lys Gly GIu Leu Val Pro Asp GIu Val Thr lie Giy lie Val Lys Giu Arg Leu Gly Lys Asp Asp Cys Glu Arg GlV Phe Leu Leu Asp GlV Phe Pro Arg Thr Val Ala Gin Ala Glu Ala Leu Gu Glu lie Leu Gu Gu Tyr Gly Lys Pro Ile Asp Tyr Val lie Asr Ile Giu Val Asp Lys Asp Val Leu Met GIu Arg Leu Thr Gly Arg Arg lie WO 2010/079357 PCT/GB2010/050018 73 Cys Ser Val Cys (y Thr Thr Tyr His Leu Val Phe Asn Pro Pro Lys Thr Pro Gly Ile Cys Asp Lys Asp (Thy Giy Glu Leu Tyr GIn Arg Ala Asp Asp Asn Gu Gu Thr Val Ser Lys Arg Leu Gu Val Asn Met Lys Gin Thr Gin Pro Leu Leu Asp Phe Tyr Ser Giu Lys Gly Tyr Leu Ala Asn Val Asn Gly Gin Gin Asp lie Gin Asp Val Tyr Ala Asp Val Lys Asp Leu Leu Giv Giv Leu Lys Lys The invention is now described in specific embodiments in the following examples and with reference to the accompanying drawings in which: Figure 1 shows activity of adenylate kinase (AK) enzymes after treatment at 70'C (A), 80*C (B) and 90'C (C): Figure 2 shows the stability of a range of AK enzymes recombinantly expressed in Ecoi. Genes encoding AK enzymes were cloned and expressed as described in Example 3. All genes were expressed from the vector pET28a except for S.acid0ocaldarius clone I which was expressed from pET3a as described previously. Expression levels were similar for each clone but a proportion of the Pyrococcus furiosus (P. fu) enzyme was in the insoluble fraction and this is likely to have reduced the amount of this enzyme being assayed. The stability of the recombinant enzymes was measured following incubation at 80'C for 30 minutes in a crude Ecoi lysate at 10 fold serial dilutions from 1 mg/ml total cellular protein (such that column 12 is equivalent to 1 fg/ml total protein). Enzymes from Thermotoga maritime and Archaeoglobus fulgidus showed significantly greater stability than the other enzymes tested, although the remaining enzymes (Suifolobus solfataricus (S. so P2), Aeropyrunm pemix and P. fu) showed similar activity to the S.acidocalcarius enzyme used as the basis of previous assays (data labelled as S. ac I); Figures 3A & 3B show the relative levels of non-reporter adenylate kinase activity (Fig 3A) and ATP (Fig 3B) in a variety of samples relevant to clinical diagnosis. Samples from healthy donors were assessed for the levels of ATP generated by non-reporter adenylate kinase (after addition of ADP as substrate; Figure 3A) or present naturally in the sample (Figure 38). This information can be used to assist in deciding which background reduction steps need to be included in assays for particular samples, although this information does not preclude their use in any assay type, particularly where infections can influence the background levels of either ATP or reporter kinase.
WO 2010/079357 PCT/GB2010/050018 74 Samples are whole blood and sera from sheep, mouse brain homogenate (MBH; representative of tissue biopsy sarnples), cows' milk, and two saliva samples (1 and 2) collected using either a citric acid ("ca") rnethod or swab device ("r"). The relative light units generated from the raw assay are converted into ATP units based on a standard Curve; Figures 4A & 4B show the differential inhibition of reporter kinase and non-reporter (endogenous) tissue kinase using Ap5a (Diadenosine pentaphosphate pentasodium salt) (Figure 4A) and the effect of Ap5a on luciferase (Figure 4B). The reporter adenylate kinases from S.I acidocaldarius or T. maritima were purified as described previously. Rabbit myokinase (muscle adenylate kinase) was obtained front Sigma. 100 ng of each enzyme was incubated with the inhibitor at the concentrations shown in reaction buffer (15 mM MgAc, 10 rnM tris, 1 mM EDTA pH 7.75) for 5 minutes. ADP was added to a final concentration of 70pM and the reaction incubated before addition of luciferin and luciferase. The RLUs gene rated following detection with luciferase/ luciferin were converted to equivalent ATP units using a standard curve and the results are shown in Figure 4A. An ICso (the concentration of inhibitor which reduces the activity of the enzyme by 50%) was calculated and gives values of 10.4 pM (Sac). 4.3 pM (Tna) and 0.06 pM (Rabbit myokinase). The presence of Ap5A does not have a detrimental effect on the activity of the luciferase (see Figure 48); Figure 5 shows the configuration of a lateral flow device for detection of an analyte in a sample; Figure 6 shows the configuration of a filtration device for the detection of an analyte in a sample; Figures 7A and 7B show the effects of further inhibitors on the background activity derived from mammalian tissues or samples and/ or background from other sources (e.g. yeast contamination). Experiments were carried out essentially as described for Figure 4. No adverse effect on the activity of luciferase was observed for any of the inhibitors examined (results not shown). Yeast adenylate kinase was obtained from Sigma. Figure 7A; comparison of inhibition of adenylate kinases by Ap6A, MAK = rabbit muscle AK (myokinase); YAK = yeast AK; SAC = S. acidocladarius AK; TMA = T. maritima AK. Figure 7B; comparison of Ap5A and Ap6A for inhibition of contaminating background adenylate kinase from either mammalian cells (MAK) or yeast (YAK). AP4A WO 2010/079357 PCT/GB2010/050018 75 (not shown) and Ap6A gives similar profiles to Ap5A for differentiating between an example of a monomeric (bacterial) reporter adenylate kinases (from Thermotoga maritima) and an exarnple of a trimeric (archaeal) adenylate kinase frorn Suffolobus acidocaldarius when either is compared to a representative example of non-reporter mammalian tissue adenylate kinase (Figure 7A). Ap4A (not shown) and Ap6A do not allow for an assay to distinguish between the bacterial and Archael enzymes and an enzyrne of fungal origin (represented here by the AK frorn Saccharomyces cerevisiae) (Figure 7B). In this case Ap5A can still be used to distinguish the reporter adenylate kinases from the yeast enzyme. Example 1 - Purification of native adenylate kinase enzymes Biomass was produced from twenty-four diverse microorganisms (Table 3). Eight members of the Archaea were represented along with sixteen diverse aerobic and anaerobic bacteria. AKs from each of these organisms were purified by affinity chromatography using selective absorption and desorption from Cibacron Blue 3A (Blue Sepharose). All enzymes were further characterised and purified by gel filtration (Superdex G200). This enabled identification of the major AK fraction and estimation of molecular mass. Table 3: List of organisms cultured to produce bioniass for isolation of AKs. Organsrn .Domain Growth To WMpHo I Aeropyrum pernix Archaeon Aerobe 951C 7.0 2 Alicyclobacilus acidocaldarius Bacterium Aerobe 650C 3.5 3 Aquifex pyrophilus Bacterium Microaerophi 5C 6.5 leeberophile 4 Bacillus caldotenax BT1 Bacterium Aerobe 651C 7.0 5 Bacillus species PS3 Bacterium Aerobe 65,c 7.0 6 Bacillus stearothermophilus 11057 Bacterium Aerobe 651C 7.0 7 Bacillus stearothermophilus 12001 Bacterium Aerobe 65,c 7.0 8 Bacillus Bacterium Aerobe 651C 7.0 9 Clostridium stercocorarium Bacterium Anaerobe 55,c 7.0 10 Meiothermus ruber Bacterium Aerobe 60C 6.5 11 Pyrococcus turiosus Archaeon Anaerobe 95,c 7.5 12 Pyrococcus horikoshii Archaeon Anaerobe 95C 7.0 13 Pyrococcus woesei Archaeon Anaerobe 95,c 7.0 14 Rhodothermus marinus Bacterium Aerobe 701C 6.5 WO 2010/079357 PCT/GB2010/050018 76 15 Silfolobus acidocaldarius 8-3 Archlaeon Aerobe 7*C 2. 16 Sijlfolobus shibatae B21 Archlaeon Aerobe 75C 2.5 17 Siulfolobus solfataricus P2 Archlaeon Aerobe 75C 2.5 18 Thermnsanaerobacter ethanoicus Bacterium Anaerobe 65,C 6.0 19 Thermoanaerobacter Bacterium Anaerobe 65,C 6.5 20 Thermobrachium celere Bacterium Anaerobe 60,C 7.0 21 Thermococcus itorais Archlaeon Anaerobe 85,C 6.5 22 Thermus aquaticus YTI Bacterium Aarobe 7*C 8.0 23 Thermus caldophius GK24 Bacterium Aerobe 70C 2.0 24 Thermus thermophilus HB8 Bacterium Aarobe 7*C 8.0 Example 2 - Analysis of stability of native adenviate kinases The stability at 70, 80 and 90*C of adenylate kinases isolated frorn biornass from organisms was assessed, and the results shown in Fig. 1. The adenylate kinases were isolated frorn the biomass by affinity chromatography using selective absorption and desorption from Cibacron Blue 3A (Blue Sepharose). The sarnples eluted frorn the columns were diluted 1:10 000 and then 1Opl of each added to a rnicrotitre well. 2.5pl of apyrase was added to each well to destroy the ATP present from the elution buffer, and incubated at 370C for 30 rninutes. The apyrase was inactivated by heat treatment at 65"C for 20 minutes. ADP substrate was added and incubated at either 70 (panel A). 80 (panel B) or 90'C (panel C) for 30 minutes and cooled to 25"C before the addition of 10pl of D-luciferin luciferase reagent. The ATP produced was measured as RLU on a plate lurninorneter. Example 3 - Expression and purification of recombinant adenviate kinases Clones expressing representative AKs were secured and recombinant AKs from the archaeon Sulfolobus acidocaldarius and the bacterium, Bacillus stearcOthermophilus produced. The plasmids were transformed into Ecoli and the cell extracts shown to contain protein bands on electrophoresis corresponding to the expected molecular masses of the AKs. AK activity was measured after incubation at the appropriate temperature (80'C for the Sulfolobus acidocaldarius AK and 60"C for the Bacillus stearothermophilJus AK).
WO 2010/079357 PCT/GB2010/050018 77 Purification methods for both AKs were established and included an initial heat treatment of incubation for 20min at 80"C, to inactivate and aggregate proteins derived from Ecoi, followed by affinity chrornatography and gel filtration. The affinity chromatography involved adsorption of the enzyme to Blue Sepharose, followed by specific elution with a low concentration of AK co-factors (AMP+ATP and magnesium ions). The ATP and AMP (Sigma) in the elution buffer were degraded by incubation with mesophile apyrase, which is readily inactivated by subsequent heat treatment. Gel filtration chromatography was scaled up to utilise a preparation grade Superdex column to enable large quantities of both enzymes to be prepared. Primers were designed for PCR amplification of the AK genes from the organisms identified during the screening of candidate native enzymes. The microorganisms were grown using individually defined growth conditions and genomic DNA isolated and used as templates for PCR amplification of the adenylate kinase genes from each organism. PCR amplified adenylate kinase genes from the organisms, Thermotoga maritima. Aerop yrum pernix, Sulfolobus acidocaldarius and Su/folobus so/fataricus were sub-cloned into the vector, pET28a and transformed into a codon enhanced Ecoi strain expressing rare tRNAs (Zdanovsky et al. 2000). This Ecoli strain is suitable for enhancing expression levels of AT-rich genes. The success of the transformation was assessed by a mini-expression study, and the results analysed by SDS-PAGE of the culture supernatants before and after induction with IPTG. SDS-PAGE was also used to analyse the supernatants after inclusion of a heat treatment step, which consisted of heating the sample to 80"C for 20 minutes prior to running on the SDS-PAGE gel to remove heat labile proteins present in the sample. Example 4 -- Analysis of the stability of recombinant adenylate kinases The stability of recombinant tAK enzymes was assessed in crude Ecoli cell lysates.
WO 2010/079357 PCT/GB2010/050018 78 Cells were grown essentially as described in Example 3 and lysed by sonication. The AK activity of the crude extract was determined both before and after heat treatment at 80"C for 30 minutes followed by 10-fold serial dilution. The results (see Figure 2) demonstrate that a wide variety of recombinant enzymes are suitable for the use in the method of the invention. Particularly preferred AKs are those from Tmarihima, A.fucidus and S.solfataricus. Such enzyrnes are likely to provide a greater dynamic range for the bioluminescent assay, if required, to provide still further sensitivity. Example 5- Genetic modification of adenylate kinases to improve stability Site-directed mutants were constructed in the AK gene frorn Pfur/osus, Phorikoshil and S.acidocadarius as shown in Examples 6-8 and SEQ IDs 17-19 respectively, using standard rnethods known to those familiar with the art. In addition to specific changes identified in each gene, the regions underlined in the S.acidocaldarius sequence form the core packing region of the archaeal adenylate kinase trimer structure. Hence amino acid substitutions that disturb the packing of this region are likely to have a najor effect in decreasing the thermal and physical stability of the enzyme. Conversely amino acid substitutions that improve the core packing, in particular hydrophobic residues with large side chains, may stabilise the enzyme to heat or other processes. Therefore in addition to the specific mutations already described a number of "selective" approaches were used with localised gene shuffling of related gene sequences in these regions (essentially as described in Stemmer (1994) Nature 370:389-391 and Crameri et al (1996) Nature Biotech. 14:315-319) and random PCR based mutagenesis using degenerate oligonucleotides or modified nucleotide mixes (e.g. Vartanian et al (1996) Nucleic Acid Res.24:2627-2633). A number of these modifications show altered stability when assessed by recombinant expression in Ecoli and rapid assay of adenylate kinase activity in lysed cells at high temperature. Example 6 - Adenylate kinases from Pyrococcus furiosus genetically engineered to provide improved stability (SEQ ID NO, 17) WO 2010/079357 PCT/GB2010/050018 79 MPFVViHTGI PGVGKSTITR LALQRTKAKF RLINFGDLMF EEAVKAGLVK HRDEMRKLPL (K TO E) IQRELQMKA AKKI (T TO A) EMAKE HPILVDTHAT IKTPHGY (M TO L) LG LPYEVKTLN PNFVHEAT PSEILGRRLR DLKRDRDVET EEQlQRHQDL NRAAAIAYAM HSNALIKHE NHEDKGLEEA VNELVKILDL AVNEYA Mutations at one or more or all of the sites indicated modify the stability of the enzyme. In addition to the three defined changes highlighted, modification of the alanine at position 157 to another small hydrophobic residue (such as i, L) or larger hydrophobic residue (such as F) increases the stability of the recombinant protein. Hence, there are 35 variants possible through combination of modifications at these sites. Modification of amino acid 157 to a polar residue such as the T (as observed at the equivalent position in AdkA of P.horikoshii), S Y, D, E, K, R results in a decrease in stability. Example 7 - Adenylate kinases from Pyrococcus horikoshii genetically engineered to provide improved stability (SEQ ID NO. 18) The modification of either or both of the residues shown in bold and underlined increases the stabilty of the enzyme (3 variants are possible). MPFVVHTGI PGVGKTIITK LALORTRAKF KLINFGDLMF EEALKLGLVK HRDEMRKLPL EVORELOMNA AKKIAEMAKN YPILLDTHAT IKTPHGYLLG LPYEVIKILN PNFIVIIEAT PSEILGRRLR DLKRDRDVET EEQIQRHODL NRAAAAAYAM HSNALKUIE NHEDKGLEEA VNELVKILDL AVKEYA Example 8 - Adenylate kinase from Sulfolobus acidocaldarius genetically engineered to provide improved stability (SEQ ID NO. 19) The modification of the underlined residues shown can increase the stability of the enzyme. MKlGIVTGIP GVGKSTVLAK VKEILDNQGI NNKIINYGDF MLATALKLGY AKDRDEMRKL SVEKQKKLQI DAAKfGIAEEA RAGGEGYLI DTHAVIRTPS GY (A TO M) PGLPSYV ITEINPSVIF LLEADPKllL SRQKRDTTRN RNDYSDESVI LETINFARYA ATASAVLAGS TVKVIVNVEG DPSIVANEI RSMK Example 9 - Expression of acetate and pyruvate kinases Following the methods of Example 3, we expressed acetate and pyruvate kinases: SEQ ID No. 20 - Acetate kinase from Thermatoga maritima SEQ ID No. 21 - Pyruvate kinase from Pyrococcus horikoshii SEQ ID No. 22 - Pyruvate kinase from Suffolobus solfataricus SEQ ID No. 23 - Pyruvate kinase from Thermoioga maritima WO 2010/079357 PCT/GB2010/050018 80 SEQ ID No. 24 - Pyruvate kinase from Pyrococcus fuiosus SEQ ID No. 25 - Acetate kinase from Methanosarcina thermophila SEQ ID No 78 - Adenylate kinase from Ecoli SEQ ID No 79 - Pyruvate kinase from Ecoli SEQ ID No 80 ~ Acetate kinase from Ecoli SEQ ID No 81 - Adenylate kinase from Methanococcus voltae (MVO) SEQ ID No 32 - Adenylate kinase from Meihanococcus thermolhotrophicus (MTH). SEQ ID No 83 ~ Adenylate kinase from Bacillus globisporus SEQ ID No 84 - Adenylate kinase from Bacillus subtilis Example 10 - Detection of Hepatitis C in an oral fluid sample Antibodies are raised against Hepatitis C surface antigens derived from either structural proteins (e.g. El and E2) or non-structural proteins (e.g. NS2, NS3, NS4A, NS4B, NS5A, NS5B) using standard methods. In brief, the proteins are expressed as either recombinant proteins in Ecoli, or synthesized as short immunogenic peptides. Short peptides are conjugated to a suitable carrier, such as HLA, and injected intramuscularly into rabbits or guinea pigs at concentrations of approximately 100pg/ml. Freund's complete adjuvant is used for the first stage of immunization, with incomplete adjuvant used subsequently. Polyclonal serum is collected after three monthly challenges over a time-course of 3 months. IgG is purified from the blood and conjugated to Tma tAK using standard coupling chemistry, In brief, the antibody is derivatised using SPDP (Pierce Chemical company) at a molar ratio of 3 SPDP to 1 Tma tAK. The free sulfhydryl in the Tma is released by limited treatment with DTT and the protein reacts with the derivitised antibody. The antibody-tAK conjugate is then separated using gel filtration chromatography. An oral crevicular fluid sample is collected using a suitable swab device. The device is heated for 1 minute at 90'C in a dry oven and then mixed with I nl of solution containing the anti-HCV polyclonal antibody-tAK conjugate. The swab is then rinsed in WO 2010/079357 PCT/GB2010/050018 81 cold water to remove any unbound conjugate and inserted into a reagent tube containing a reagent mix comprising Mg-ADP, luciferin and luciferase. The swab is incubated for 2 minutes and then the entire reagent tube is inserted into a hand-held hygiene monitor and the read-out measured immediately. Example 11 - Detection of immune status in a sample of serum or whole blood e.. following immunisation with measles vaccine or at an early stage following exposure to infectious measles virus A fragment of the measles glycoprotein, other measles virus surface components or heat inactivated measles virus. is used to coat a solid support, such as a dipstick. A sample of whole blood, diluted 1:2 with PBS including up to 2M urea to inactivate any non-reporter kinase is applied to the dipstick and antibodies against the measles components are allowed to bind (binding step 1; 5 minutes at 30cC). Apyrase is added to the blood sample to inactivate any ATP during this phase. After brief rinsing with phosphate buffered saline (PBS; pH7.4), the dipstick is imrnersed in a solution containing anti-human lgG conjugated to tAK and incubated (binding step 2: 5 minutes at 30 0 C). Again the dipstick is rinsed briefly and then placed within a reagent tube. Luciferin/ luciferase and ADP were added simultaneously and the reaction measured using a hand held luminometer after 5 minutes. Example 12 - Sample preparation for detection of norovirus in stool samples Norovirus is routinely measured in diarrheal samples (i.e. stool sample) for the purposes of clinical diagnosis. To reduce the levels of contaminating kinase activity the stool sample is diluted between 1:2 and 1:4 with a buffer designed to inactivate the contaminating kinase. This buffer includes one or more of the following components: 2M urea; 2M guanidine; 1 % SDS; 1% deoxycholate; 1% Triton X1 00 The addition of the above components also makes the norovirus antigen more readily detectable by the antibody conjugates described in the next example, increasing the WO 2010/079357 PCT/GB2010/050018 82 assay signal as well as reducing assay noise. Optionally, apyrase may also be added to the sample destroy any ATP that may be present. The same types of additive can also be used as sample processing components for the detection of norovirus in vomitus, a sample which would be useful to test for norovirus but which has not, to date, been suitable for analysis. Example 13 - Lateral flow assay for the detection of norovirus andlor C-difficile toxin in a stool sample A reporter kinase conjugate is prepared by conjugating the adenylate kinase from P.abvssi to norovirus VP1 protein or fragments thereof (e.g. the P-domain (located between amino acids 362 and 703), the P2 domain (amino acids 414-589). or sub fragments of the Pl domain (aa 362-413 or 590~703). The positions within the norovirus correspond to the numbering as described in Chen R, Neill JD, Estes MK, Prasad BV. X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity. Proc Natl Acad Sci U S A. (2006)103 p8048-53. A lateral flow device is prepared essentially as shown in Figure 5. The sample-receiving zone is coated with an anti-norovirus antibody or antibodies (to provide detection of the antigenically diverse range of clinical isolates). The reporter kinase conjugate (described above) is then bound to the sample-receiving zone via the antibodies. The clinical stool sample is processed as outlined in Example 12 above and applied to the sample-receiving zone of the device. In the presence of norovirus, the reporter kinase conjugate is displaced and migrates to the detection zone, via the background reduction zone. The background-reduction zone comprises an anion exchange membrane which retains any ATP contained within the original sample. By using a buffer at neutral pH (such as PBS) the ATP is retained on the anion exchange membrane whilst the reporter kinase conjugate passes through as it remain below the isoelectric point and is therefore cationic. Non-reporter kinase has previously been removed in the sample preparation phase (see Example 12).
WO 2010/079357 PCT/GB2010/050018 83 The lateral flow device is then snapped in two and the detection zone is then placed into a reagent tube containing ADP, luciferin and luciferase. The presence of norovirus in the original sample is determined by measurement of light output with an assay tirne of 2-5 minutes. Similarly a lateral flow device may be provided to detect the presence of C.difficile toxin A or toxin B in a sample. Antibodies to these targets are well described in the literature and can be conjugated to reporter adenylate kinase(s) as described above. The stool sample is processed as in example 12 and the lateral flow assay carried out as described. Optionally a device may be provided to detect the presence of either C. difficile toxins) or norovirus in a sample, enabling differential diagnosis of clinical samples to be carried out. The sample is processed as described in example 12 and mixed with diagnostic reagents for both norovirus and C. difficile toxin(s) in the same reaction. The sample may be run on two separate lateral flow devices set up to capture only one of the two targets or preferentially on a single device with two capture windows. These two devices or two windows are then assayed separately to determine the presence of one or more of the target species. Example 14 -Detection of legionella in a water sample The assay is carried out using a device as set out in Figure 6. A water sample from a cooling tower is sampled at the point of routine maintenance. Typically 50ml of water is added to a syringe which already contains latex beads coated with anti-legionella antibody (antibody A; or fragment thereof) and the reporter kinase from A. fuigidus chemically conjugated to a second anti-legionella antibody (antibody B), Optionally antibodies A and B may be the same antibody provided there are multiple binding sites on the surface of legionella. Preferably they are different antibodies recognising different e pitopes of the legionella. If legionella is present in the water sample, it becomes bound to the latex bead via antibody A. The reporter kinase is WO 2010/079357 PCT/GB2010/050018 84 bound to the latex bead via the interaction of antibody B with the already-bound legionella. The syringe is shaken continuously for 5 minutes either by hand or optionally within a suitable automated shaker. The syringe is applied to a filtration device which contains a filter designed to allow the free passage of the water, non-reporter kinase, ATP, uncomplexed reporter kinase conjugate, and any uncornplexed microorganisms, but which will retain anything bound to the latex bead. Thus, any reporter kinase bound to the latex bead will be retained on the filter. The filter is removed from the filter housing and transferred into a reagent tube. The presence of legionella is assessed by the addition of ADP, luciferin and luciferase and the measurement of light output using a portable luminometer. Example 15 - Detection of Chlamydia in a swab sample A swab device is used to collect a vaginal sample from the test individual. The swab is placed in a reagent tube that contains 1M urea to assist in disrupting the tissue and 2pM Ap5A final concentration which blocks the activity of any non-reporter kinase. The presence of Ap5A does not have a detrimental effect on the activity of the luciferase (see Figure 48), hence even if it is present in the final reaction mixture it does not adversely affect the limits of detection. A reporter kinase conjugate is prepared by conjugating the adenylate kinase from S. solfatai'cus to a Chlamydia antigen. A suitable Chlamydia antigen is the major outer membrane protein (MoMP) which is present in high copies on the surface of Chlamydia. A series of polymorphic membrane proteins have also been described and may represent suitable target antigens for specific and sensitive detection. Antibodies can be generated to this protein, or peptides derived from it according to conventional protocols. A lateral flow device is prepared as set out in Figure 5. The sample-receiving zone of the device is coated with an antibody to a Chlamydia antigen, The reporter kinase conjugate WO 2010/079357 PCT/GB2010/050018 85 is then applied onto the sarmple-receiving zone of the device, and becomes attached thereto via the interaction between the antigen of the conjugate and the coated antibody. A small volume of the sample is then spotted onto the sample-receiving zone of the device. Any chlamydia antigen present in the sample displaces the reporter kinase conjugate from the sample-receiving zone and allows flow of the reporter kinase conjugate to the detection zone where it can be measured. The device is then placed in a reagent tube, and with ADP and luciferin/ luciferase reagents. The light output signal is measured within 5 minutes. As an alternative antigen, antibodies raised to the bacterial lipopolysaccharide from Chlamydia may be employed and conjugated to the reporter kinase. This multivalent target may provide greater sensitivity and specificity than other targets. Optionally more than one of the target antigens may be combined to amplify the signal detected. Example 16 - Detection of Listeria in a food sample A food sample suspected of containing Listeria is immobilized onto a microtitre plate by non-specifically binding sample components to the plate, treating the plate to prevent further non-specific binding thereto and washing. A reporter kinase conjugate is prepared by conjugating an antibody specific to Listeria to the pyruvate kinase from S. solfataricus. The reporter kinase conjugate is applied to the plate and allowed to bind, prior to further washing/recovery. The plate is now heated to about 90C for about 1 minute in a cell extraction buffer (in a thermal cycler) to denature any non-reporter AK present and release any ATP that may be trapped within the micro-organism. The plate is then cooled to 37"C and a thermolabile ATPase such as apyrase added. The plate is incubated for about 5 minutes to remove the background ATP, then the temperatures is raised to about 90QC to denature the thermolabile ATPase.
WO 2010/079357 PCT/GB2010/050018 86 Next, ADP and a mixture of luciferin and luciferase mixture are added simultaneously to the plate. The kinase acts on the ADP to generate ATP, which subsequently reacts with the luciferin / luciferase to produce light. The light output is measured using a hand-held luminometer and is directly proportional to the concentration of the microorganism present. Example 17 - Detection of Salmonella in a food sample A solid phase is prepared by coating magnetic beads with a first anti-salrnonella polyclonal antibody raised in Guinea pig. A reporter kinase conjugate is prepared by conjugating the adenylate kinase from T. maritima to a second anti-salmonella polyclonal antibody raised in Guinea pig. The food sample to be tested is dispersed in a buffer containing 1 M urea plus 2pM Ap5A and mixed for 5 minutes, in the presence of the magnetic beads and the reporter kinase conjugate. This mixing can be carried out at either room temperature or an elevated temperature. If Salmonella is present in the food sample, it will bind to the first anti salmonella antibody on the magnetic bead. In turn, the reporter kinase conjugate will bind to the magnetic bead via the interaction between the second anti-salmonella antibody and the already-bound salmonella. The magnetic beads are then collected by attraction to a strong magnet and washed with a neutral buffer. The magnet with beads attached is transferred to a reagent tube and ADP, luciferin and luciferase are added simultaneously. The light output signal is read in a luminometer, preferably hand-held, within 5 minutes. Example 18 - Validation of processes for sterilising bulk liquids Preparation of Indicator I A first indicator is prepared by covalently attaching 0.1mg of pyruvate kinase from Sulfolobus solfataricus to a polystyrene strip. Preparation of Indicator 2 WO 2010/079357 PCT/GB2010/050018 87 A second indicator is prepared by attaching 0.1mg of the adenylate kinase from A.fulgidus to the inner face of a semi-permeable membrane such as a dialysis tube. The A.fulgidus kinase contains a naturally occurring reactive cysteine residue (iKe. not disulfide-bonded within the native enzyme), which can be reacted with BMPH (Pierce). This generates a group capable of reacting with oxidised carbohydrates, as generated, for example, by the treatment of Visking tubing with a suitable oxidising agent. The enzyme is reacted with the oxidised rnembrane surface to generate a covalently linked indicator. Validation The indicator is then attached within the bulk liquid and the sterilisation process (such as autoclaving, the passage of oxidative gases or other chemical sterilisation) is carried out. The indicator is removed from the bulk liquid on completion of the process, and the residual activity of the kinase is measured. To achieve the measurement the indicators are first incubated in the presence of apyrase, at a concentration of 10pg/ml for 2 minutes. The apyrase can be inactivated by addition of Ap5A at a concentration of 5pM. The two indicators can then be read independently by addition of a combined reagent containing ADP, luciferin and luciferase. The measurement is made within 5 minutes using a hand held luminometer, such as a hygiene monitor. In this example any non-reporter kinase that might be present is destroyed by the treatment conditions and as such specific kinase-reduction steps are not required. The residual activity is then compared to a defined threshold value. Example 19- Validation of the performance of cloth washing cycles using biological detergents Preparation of Indicator I A first indicator is prepared by cross-linking a adenylate kinase from Ssoifatarcus onto a flexible polystyrene wand using a method based on disulfide bond formation. In this method, the adenylate kinase is derivitised with a heterobifunctional agent such as WO 2010/079357 PCT/GB2010/050018 88 Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]- propionamido)hexanoate (SPDP: Pierce chemical company, UK) at a ratio of between 1-3 SPDP:protein. The derivatised kinase is then reduced by reaction with a reducing agent such as dithiothreitol (DTT), or 2 mercaptoethanesulfonic acid (MESNA), the reducing agent removed by dialysis. and the kinase reacted with a maleirnide-derivatised polystyrene surface. Typically, 0.1 mg of kinase is present on the indicator. Preparation of Indicator 2 A second indicator is prepared by the non-specific adherence of an adenylate kinase from S.acidocaidarius onto a high-protein binding polystyrene strip. The kinase is prepared at a concentration of 0.5-2 mg/ml in a bicarbonate buffer (pH 9.6), optionally containing the stabilising agent sorbitol at between 0.1 and 2% w/v. The kinase in binding buffer is then incubated with the high protein-binding polystyrene strip for a period of 1-2 hours at 224C (or 40C overnight). The residual kinase is removed by washing in a phosphate buffered saline. Typically, 0.1 mg of kinase is present on the indicator. Validation of wash cycles The washer is loaded with the terns to be washed, and the indicator is fixed within a suitable holder on the inside of a washer (to facilitate its recovery). The wash cycle is then performed. At completion of the cycle, the indicator is removed and the residual activity of the kinase is assessed. in this example the washing process removes and/or inactivates both any non-reporter kinase and any residual ATP, hence neither interfere with the assay. The presence of the reporter kinase is determined by the addition of ADP, followed within 1 minute by the addition of luciferin and luciferase. If the measurement of residual kinase activity is equal to or below a predetermined threshold level, then the load is cleared for further processing. Examrple 20 - Preparation of a fibrin-based indicator device Preparation of tAK fusions for cross-finking to fibrin WO 2010/079357 PCT/GB2010/050018 89 A transglutaminase substrate sequence (MNOEOVSPLGG - SEQ ID No: 33) is added on to the Nterminus. the C-terrninus., or both N- and C-termini, of the adenylate kinase from S. acidocaldkrius encoded by a codon optirnised gene clone. This construct is transferred as an Ndel - Sall fragment into an in-house expression vector (pMTL 1015; as described in WO 2005/123764). The expression construct is confirmed by DNA sequencing and transferred into expressions hosts BL21 or RV308 for subsequent expression. Similarly, the resynthesised tAK gene from Thermatoga maritime (SEQ ID 29) is fused to the transglutaminase sequence in the three orientations identified above. The cloning and preparation of the expression system is also as described above. The fusion constructs can also be expressed in other expression vector-host combinations with the addition of affinity tags for subsequent purification. Particularly useful in this context are expression vectors which add 6-histidine tags on either the N or C-terrninus of the fusion proteins, modifications which aid purification and detection but do not interfere with the intrinsic properties of the fusion proteins. Vectors for this type of modification include pET series vectors (Novagen / Merck) and pQE series vectors (Qiagen). To generate material for the indicator devices the expression strains are grown initially in 8-litre fermenters essentially under static culture conditions. In brief, the strains are prepared as seed stocks and subsequently diluted into the 8-litres of growth media (modified terrific broth containing additional glucose). The cultures are grown under standard fermentation conditions until the cultures reached an optical density (OD at 600nm) demonstrating that they are entering stationary conditions (typically at around an OD = 5). The fermenters are then held under minimally aerated conditions for up to 12 hours prior to harvesting of material by continual centrifugation. Purification of tAK fusions The harvested material is then purified according to the following protocol.
WO 2010/079357 PCT/GB2010/050018 90 Buffer A: 20mM Tis-HCI; 900mM NaCl, pH 7.5 Wash Buffer: 20mM Tris-HCI; 200mM NaCl, pH 7.5 Buffer B: 20mM Tris-HC; 200rnM NaCL pH 7.5 10mM ATP; 10mM AMP; 10mM MgCl2 MgAc buffer: 15mM MgAc (1M Fluka BioChemika), pH 6.8 1. Weigh frozen cell paste (10g) and resuspend in 3x (30rnl) volurne of Buffer A, pH 7.5. 2. Sonicate on ice (-12,000khz) using 25 cycles of 30 seconds on / 30 seconds off. Take I ml sample. 3. Sonicated cell solution is centrifuged at 6,000rpm for 30mins at 4 degrees C. Supernatant carefully poured off and 1 mi sample taken. 4. Supernatant is heat treated at 80 degrees C in a water bath for 20 mins. 1 ml sample taken. (This step is an optional step depending on thermal stability of the fusion proteins). 5. Heat treated solution centrifuged at 6000 rpm for 30 mins at 4 degrees C. Pour off supernatant and take 1 ml sample. 6. Filter the sample with 0.2 pm low binding filter before loading onto column. 7. Equilibrate Blue Sepharose Fast Flow column with 5 Column Volumes (CVs) of Buffer A. 8. Load the sample. VVash column with wash buffer at 0.2 mi/min overnight. 9. Elute protein with 100% buffer B at a flow rate of 1 ml/min collect product in 2.5 ml fractions. 10.Once all proteins have eluted wash column with 100% buffer B at 5 ml/min for 5 CV's. 11. Re-equilibrate column with 5 CV's buffer A. 12. Rinse column with 5 CV's 20% Ethanol for storage at 4OC. Optionally, additional protein purification methods are applied to yield a higher purity product. Ion exchange chromatography on either SP-Sepharose Fast Flow or Q Sepharose Fast Flow resins is pariicularly effective.
WO 2010/079357 PCT/GB2010/050018 91 The samples are then analysed using a standard assay format to identify fractions containing peak adenylate kinase activity. This is confirmed by SDS-PAGE analysis using standard techniques. In brief, the assay method is carried out using the following protocol: 1. Dilute the purified tAK fusion 1:1000 and 1:10,000 in Mg Ac Buffer. Add 100 pi per well 2. Treat with Apyrase (50 pl /well at 2.5 units per ml stock concentration; Sigma Grade VI Apyrase from potato) and incubate for 30 mins at 30*C. with shaking. to remove ATP. 3. Incubate plate at 70'C for 10 mins to denature Apyrase. 4. Add 50pl/well of ADP (275 pM ADP in MgAc buffer) and seal. Incubate at 70*C for 20 mins. 5. Remove plate and allow to cool to room temperature for 20 mins, warm Luciferase / Luciferin (L/L) reagent to room temperature for 20 mins. 6. Add 200 pi ATP standard to 1 or 2 empty wells per plate. 7. Set up injectors on luminometer and prime them with L/L reagent (ATP reagent, Biotherma). Inject 30 pl L/L reagent /well. 8. Read light generated immediately using luminorneter. The fractions with peak kinase activity are then dialysed extensively against phosphate buffered saline (PBS pH 7.4) and stored until required. Optionally a fusion can be prepared between tAK and the full length fibrinogen molecule to provide further means to incorporate the enzymatic activity within the fibrin film. Deposition of tAK fusions onto a solid support The tAK-fibrin fusion is diluted to around 200 pg/ml in either PBS or bicarbonate buffer (pH 9.6) and applied to a solid support of 316L grade stainless steel, plastic, glass or textiles. The protein is allowed to adhere to the surface for up to 2 hours at room temperature or overnight at 4'C.
WO 2010/079357 PCT/GB2010/050018 92 Optionally, additional carrier molecules are added at this stage, e.g. sucrose at concentrations up to 1 % w/v, albumin at up 1 mg/ml, pig rnucin at up to 0.5% w/v. The addition of such carriers may be particularly important for certain types of indicator but the presence of the carrier should not interfere with subsequent interaction and cross linking to the fibrin film applied in the next stage. Overlay of fibrin-containing soil and cross-linking to fibrin-tAK fusion A solution containing fibrinogen is added to effect the cross-linking of the indicator to the fibrin-containing test soil (biological matrix). A solution containing up to 3 mg/mi fibrinogen (containing Factor XIll), 2.5 mM CaCl., and thrombin (up to 5 NIH units per ml) is mixed freshly and added to the coated surface of the solid support. The reaction is allowed to proceed at room temperature for up to 30 minutes, depending on the level of cross-linking required. Optionally, albumin (up to 80 mg/ml) and haemoglobin (up to 80 mg/mi) are added at this stage to provide a tougher and more realistic challenge for cleaning of a blood-like soil. After cross-linking, residual liquid is removed and the indicator device left to dry. Optionally, the tAK-fibrin peptide fusion is added to the fibrin-containing test soil solution (biological matrix) prior its addition to the solid support surface. Cross-linking of the fibrin peptide to the matrix can be increased by adding more Factor X111 and/or extending the duration of the reaction. Cross-linking can also be enhanced by the use of the tAK fusion protein with fibrin peptides added to both ends of the molecule. Optionally a fibrinogen tAK fusion could be added directly to this solution to provide further cross linkage of the indicator. Covalent chemical crossiinking of tAK to fibrin or fibrinogen. tAK may be chemically joined to fibrin, fibrin peptides or fibrinogen by a wide range of methods familiar to those working in the field. For example purified protein preparations for fibrinogen or fibrin are obtained from commercial sources (e.g. Sigma). The tAK from S.acidocaldarius is prepared as described above. The tAK is derivatised using the amide reactive reagent SPDP (SPDP (N -Succinimidyl 3-(2-pyridvldithio)-propionate; WO 2010/079357 PCT/GB2010/050018 93 Pierce chemical company) according to the manufacturer's instructions. The fibrin or fibrinogen is also derivatised using the same protocol. The derivatised tAK is reduced by reaction with mercaptoethanol to yield a reactive sulfhydryl group. This is then mixed with the SPDP-derivatised fibrin causing the formation of covalent bonds between the two molecules. The concentrations of the reaction partners should be determined empirically following the guidelines within the manufacturer's instructions for SPDP. The chemically linked tAK-fibrin or fibrinogen can be used interchangeably or in addition to the fusion protein. Uses of fibrinAtAK indicators Use in a washer disinfector An indicator is prepared as described above. Preferably the solid support is a rectangular stainless steel strip 55mm x 5mm x 0.75mm, which may be coated on one or both surfaces. One or preferably several indicator strips are positioned within the chamber of the washer disinfector. Optimally these may be positioned in sites which may be the most difficult to clean, providing the highest degree of certainty that the wash process has been effective. Alternatively they may be positioned to monitor the function of multiple spray arms (i.e. where these may be independent of each other). The indicator strips are clipped to the shelves or other substructure of the washer-disinfector chamber to ensure that they do not move during the wash treatment. The orientation of the surrogate devices can be modified to provide further information about the efficacy of the wash process, for example by positioning them so that the coated surface are at right angles to the direction of water spray. The instrument load is added and the standard run cycle performed. At the end of the run the devices are removed from the chamber and the presence of residual tAK-fusion assessed. as outlined below, prior to the removal of the instruments and any subsequent processing. Optionally devices can be removed during the wash process either by interrupting the process at carefully defined points or by using a machine that provides a method of withdrawing the indicator during the run. Use in endoscope test procedure WO 2010/079357 PCT/GB2010/050018 94 The indicator device for monitoring an endoscope reprocessing systern is essentially similar to that outlined above. A sirnilar size indicator surface, representative of either the stainless steel components within an endoscope, the PTFE tubing or other relevant materials is placed within a tubular chamber. This is attached, via suitable screw, push or bayonet fittings to either the front end of the endoscope or, more preferably the end which makes contact with patient tissues. This is placed within the endoscope reprocessing unit and the ends of the endoscope tubing and indicator device are coupled to the ports in the unit. The process is run as standard and the indicator device removed at the end of the run for analysis, prior to onward processing or the return of the endoscope to use. Means of assessing cleaning performance The indicator device is removed at the end of the test process. The indicator strip is then placed into a reagent tube with ADP, luciferin and luciferase, added simultaneously, with signal being read-out on a hand-held luminometer with 2 minutes. Example 21 --Preparation of tAK~Sup35 fusion Clones containing the N-terminal domain of Sup35 from Saccharomyces ceewsae fused to either the N- or C-terminus, or both termini, of adenylate kinases from either S.acidocadarius or Tmaritima are generated by standard DNA manipulation techniques. All clones are transferred as Ndel - Sall fragments into the pMTL1015 expression vector and their sequences verified. The expression constructs are used to transform BL21 or RV308 expression strains and the material grown in large scale fermentation conditions, but with minimal aeration. Expression and purification of a tAK-Sup35 fusion is essentially the same as for the fibrin-peptide fusions described in Example 20, except that the use of the thermal denaturation step (Step 4) is not part of the purification protocol. In brief, cell paste from the fermenter is resuspended in buffer A, and lysed by sonication. The cell debris is removed (no heat treatment is typically used for these type of fusions) and the supernatant used for column purification as outlined in Example 20.
WO 2010/079357 PCT/GB2010/050018 95 Under certain growth conditions the fusion proteins may be insoluble, being apparent as inclusion bodies within the cells. In this case the cell pellets are prepared and lysed in the same way, but the resulting insoluble fraction, containing the inclusion bodies, is collected by centrifugation. This rnaterial is washed in a buffer (e.g. PBS) containing Triton X100 (up to concentrations of 5%), After each wash the pellet containing the fusion proteins is separated by centrifugation. After 5 washes the inclusion bodies are resolubilised in PBS containing 8M urea and agitated gently for up to 30 minutes. Any residual insoluble material is removed by centrifugation. The urea-solubilised rnaterial is dialysed against up to 5 x 10 volumes of PBS to remove the urea and allow the fusion proteins to refold. Optionally the urea may be removed more rapidly by spraying the urea-solubilised preparation through a fine gauge needle into 100 volumes of rapidly stirred PBS or buffer A as used for purification. The material is allowed to stand at room temperature with stirring for up to 30 minutes prior to subsequent processing. Subsequent purification of the fusions is carried out essentially as described in Example 20. The supernatant from either lysed cells or solubilised and refolded inclusion bodies is loaded onto a pre-equilibrated Blue Sepharose Fast Flow column. After extensive washing in buffer A and subsequently in wash buffer, the protein is eluted using buffer B. Peak fractions are determined by SDS-PAGE analysis and enzyme assay. Fractions are then pooled and dialysed into PBS. Conversion of tAK-Sup35 to an amyloid form The Sup35-tAK fusions when assembled into fibrils are more representative of amyloid proteins such as prions which are key molecules against which to assess the efficacy of decontamination processes. The amyloid form of the Sup3-5tAK fusions is generated by either refolding of the purified soluble protein or by modifying the conditions used for dialysis of the urea resolubilised inclusion body preparations, In the first case, a conformational change is induced by exposure of the fusion proteins to conditions around pH4 (e.g by dialysis into a suitably buffered solution at pH 7.4 optionally containing up to 1M NaCl). In the latter case, the resolubilised fusion proteins in 8M urea / PBS are dialysed for 6-12 hours at WO 2010/079357 PCT/GB2010/050018 96 room temperature against 2M urea, 300mM NaCl, in PBS (pH 7.4). Alternatively, the fibrilisation can be induced by dialysis against 20mM Tris pH8.0 10mM EDTA under similar incubation conditions. Optionally, the fusion proteins may be incorporated into fibrils containing normal Sup35. This is achieved by mixing the fusions with unfused Sup35 expressed in the same way, at ratios between 1:1 to 1:10 fusion:Sup35. Deposition of tAK-Sup35 fusions onto solid support. Deposition of the fibrils onto a solid support is effected by simple protein adsorption in a suitable buffer (e.g. PBS pH 7.4 Bicarbonate buffer pH 9.6) in the presence of high levels of NaCl. The use of charged or precoated surfaces (e.g. plastics coated with Poly L-lysine) is useful in providing surfaces which can more effectively bind the fusion proteins. Optionally, the fibrils may be deposited in a suitable carrier, such as sucrose (to 1%), pig mucin (up to 0.5%), or albumin (up to 1rng/ml). Overlay of test soil A test soil (biological matrix) is then overlaid onto the amyloid preparation adhered onto the surface as described above. Suitable biological matrices in which the amyloid indicator is embedded include e.g. 0.5 % mucin, with or without albumin, a commercial test soil (such as that manufactured by Browne's) or any one of the test soils identified in guidance documents issues by national and international standards committees (e.g. Edinburgh soil as detailed in HTM 01/01 (UK). Assembly of amyloid fibrils within the test soil Given the ability of amyloids to self-assemble in complex matrices it is possible for the amyloid-tAK fusion to be mixed with soil components prior to fibril formation and subsequent deposit onto surfaces. This provides further options for indicators in which the amyloid fibrils may be mixed and inter-chelated with other soil components providing a different type of matrix that may be harder to remove from surfaces.
WO 2010/079357 PCT/GB2010/050018 97 Use of tAK-Sup35 indicator for assessing prion removal from surfaces in a washing process An indicator as described above is prepared as fibrils arid dried down onto a steel surface in the presence of 0.5% mucin. The indicator is placed within the chamber of a washer disinfector at pre-determined locations. The instrument load is added. The process is started as per the manufacturer's instructions and any process records completed. At the end of the process. and before any instruments are taken from the machine, the indicator devices are removed and assessed as described in Example 20. Use of tAK-Sup35 indicator for assessing prion inactivation in a protease-based process Indicators as described above are prepared as fibrils with a high ratio of free Sup35:Sup35-tAK (in excess of 5:1) and deposited onto solid support strips in the presence of Edinburgh soil. The indicator devices are inserted into a pre-soak bath containing freshly made Prionzyme
T
" (Genencor International) prion inactivation treatment (at 60*C, pH 12). The indicator strips are clipped to the side of the bath such that the ends of the indicators are within the bulk of the liquid. Instruments are added as required and processed for 30 minutes. The indicator devices are removed from the bath at the end of the process, prior to removal of the instruments and assessed as described in Example 20. Use of tAK-Sup35 indicator for an oxidative process aimed at destroying prions. An indicator as described above is prepared as fibrils using only Sup35-tAK, and deposited onto a stainless steel surface (optionally in the presence of 0.1% wiv sucrose). The indicator is attached to the inside of the lid of a Genesis"" container in which the instruments are prepared for processing and the lid closed. The container is inserted into the load chamber of a suitable processor for oxidative challenge (e.g. the 125L ozone steriliser; TSO 3 or a vapour phase hydrogen peroxide technology such as that described in published papers by Fichet et al 2004; Lancet) and the process run according to manufacturers' instructions. At the end of the process, the Genesis container is taken out of the chamber and the indicator devices are removed and processed as described in Example 20.
WO 2010/079357 PCT/GB2010/050018 98 Example 22 - Detection of a reporter kinase in a sample due to an infection; use for rapid assay of infection in patient sample A patient presented at the clinic with suspected infection from the obligate intracellular pathogen Burkholderia pseudomallei. A blood sample was removed and dispersed in a buffer containing 1 M urea plus 5pM Ap4A. The sample was assayed by addition of ADP and luciferin / luciferase reagent, incubated for 2 rninutes and the light output measured in a hand-held luminometer. The signal generated is directly proportional to the amount of B. pseudomallei within the blood sample. Detection of a reporter kinase in a sample due to an infection; use for rapid assay of infection in cell models The study of intracellular bacterial pathogens is complicated by the need to grow them in mammalian cell culture systems. Measurement of viable cells the requires subsequent culture or re-infection into mammalian host cells. both of which are very time consuming methods. A rapid assay, such as provided by the invention, is invaluable in providing information that can be used in real-time to determine the results of an experiment. A suitable isolate of B. pseudomallel was incubated with a permissive cell culture model capable of supporting growth of the bacteria within the cell. The culture was grown for an appropriate length of time to establish the infection. Cells were isolated by centrifugation and lysed by resuspension in a buffer containing 1% Triton X-100, 5pM Ap4A. A detection reagent containing ADP, luciferin and luciferase was added and incubated for 5 minutes. The light output was read in a 96-well plate luminometer. The amount of signal generated is proportional to the number of viable B.psudoralle cels within the cell culture. Optionally this rapid assay method can be extended to measure the effects of vaccines or drugs that reduce the number of viable cells within the cell culture. For example, antibodies raised in a patient immunised with a prototype Bpseudomallei vaccine are mixed with the organisms prior to addition to the permissive cell culture.
WO 2010/079357 PCT/GB2010/050018 99 After an incubation period sufficient to allow uptake of non-neutralised microrganisms, the cell are washed and incubated for a period of time sufficient to establish the infection. Cells are then washed and lysed as described above, again in the presence of inhibitor. The signal measured by simultaneous addition of ADP, luciferin and luciferase is proportional to the number of nonneutralised microorganisms giving a measure of the effectiveness of the vaccine and/or the immune response generated in a vaccinated individual. Such methods are suitable for high-throughput screening. In a further example, infected cell cultures are set up as described above. After the infection is established, antibiotics are used to treat the infected culture, with the express aim of killing the bacteria within the host cells. After the antibiotics have been applied, the cultures are incubated for sufficient time for the antibiotic to have its effect. The cells are lysed in the presence of inhibitor as described above and the number of viable cells quantified by measurement of the reporter kinase, by addition of ADP, luciferin and luciferase.

权利要求:
Claims (26)
[1] 1. An assay for detecting the activity of a reporter kinase. comprising: (i) adding said reporter kinase to an assay mixture, wherein said reporter kinase is contacted with ADP, and, no more than 5 minutes after being contacted with the ADP, said reporter kinase is contacted with a bioluminescent reagent. wherein, prior to contacting the reporter kinase with ADP, the assay mixture is substantially free from non-reporter kinase (ie. kinase other than reporter kinase); and (ii) detecting light output from the assay mixture.
[2] 2. An assay according to Claim 1, wherein the reporter kinase is contacted with the bioluminescent reagent no more than 2 minutes after being contacted with the ADP.
[3] 3. An assay according to Claim 1 or 2 wherein, the reporter kinase is contacted simultaneously with the ADP and the bioluminescent reagent.
[4] 4. An assay according to any previous claim, wherein, prior to contacting the reporter kinase with the ADP, the assay mixture is substantially free from ATP.
[5] 5. An assay according to any previous claim, wherein, prior to contacting the reporter kinase with the ADP, non-reporter kinase is substantially removed or inactivated.
[6] 6. An assay according to any previous claim, wherein, prior to contacting the reporter kinase with the ADP, ATP is substantially removed.
[7] 7. An assay according to any previous claim, wherein the reporter kinase is an adenylate kinase, preferably a trimeric or monomeric adenylate kinase. WO 2010/079357 PCT/GB2010/050018 101
[8] 8. An assay according to any previous claim, wherein the reporter kinase is a thermostable kinase.
[9] 9. An assay according to any previous claim, wherein the assay is completed in less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, less than 1 minute, or less than 30 seconds.
[10] 10. A method for determining the presence of an analyte in a sample, comprising: (i) exposing the sample to a reporter kinase coupled to a binding agent specific for the analyte, so that a complex is formed between the reporter kinase and said analyte when present in the sample; (ii) separating complexed reporter kinase from uncomplexed reporter kinase; and (iii) measuring the activity of the complexed reporter kinase using an assay according to any of Claims 1-9.
[11] 11. A method for determining the presence of an analyte in a sample, comprising: (i) providing a solid support comprising a reporter kinase, wherein the reporter kinase is attached to the solid support via a linker that comprises a binding agent specific for the analyte; (ii) applying the sample to the solid support, whereby said analyte when present in the sample displaces reporter kinase from the solid support; and (iii) measuring the activity of the displaced reporter kinase using an assay according to any of Claims 1-9.
[12] 12. A method for detecting the presence of an analyte in a sample comprising: (i) providing a solid support on which is attached a first binding agent specific for the analyte; (ii) exposing the solid support to the sample so that said analyte when present in the sample becomes attached to the solid support via said first binding agent; (iii) exposing the solid support to a reporter kinase coupled to a second binding agent specific for the analyte, so that the reporter kinase becomes attached to the solid WO 2010/079357 PCT/GB2010/050018 102 support via the interaction between the second binding agent and the already bound analyte; (iv) applying the mixture obtained in step (iii) to a filter membrane, wherein the solid support is retained on the filter membrane; and (v) measuring the activity of the retained reporter kinase using an assay according to any of Claims 1-9.
[13] 13. A method for detecting the presence of an analyte in a sample comprising: (i) providing a magnetic solid support on which is attached a first binding agent specific for the analyte; (ii) exposing the solid support to the sample so that said analyte when present in the sample becomes attached to the solid support via said first binding agent (iii) exposing the solid support to a reporter kinase coupled to a second binding agent specific for the analyte, so that the reporter kinase becomes attached to the solid support via the interaction between the second binding agent and the already-bound analyte; (iv) exposing the mixture obtained in step (iii) to a magnet, wherein the solid support is retained on the magnet; and (v) measuring the activity of the retained reporter kinase using an assay according to any of Claims 1-9.
[14] 14. A method according to Claim 12 or 13 wherein the solid support is selected from the group consisting of a latex bead and a magnetic bead.
[15] 15. A method of validating a treatment process for reducing the amount or activity of a contaminating biological agent in a sample, comprising the steps of: (i) providing a sample that contains, or is suspected to contain, a contaminating biological agent (ii) subjecting the sample to a treatment process in the presence of a defined amount of a reporter kinase, wherein the reporter kinase and the contaminating biological agent are both exposed to the treatment process; WO 2010/079357 PCT/GB2010/050018 103 (iii) measuring the residual activity of the reporter kinase using an assay according to any of Claims 1-9; and (iv) comparing said residual activity to a predetermined kinase activity. wherein the pre-determined kinase activity corresponds to a confirmed reduction in the amount or activity of the contaminating biological agent under the same conditions.
[16] 16. A method according to any of Claims 10 to 15, wherein the rnethod is completed in less than 15 minutes, less than 10 minutes, less than 5 minutes, or less than 2 minutes.
[17] 17. A method according to any of Claims 10 to 16, wherein, prior to measuring the activity of the reporter kinase, the sample is treated to substantially remove ATP and! or to substantially remove or inhibit non-reporter kinase.
[18] 18. A device for detecting the activity of a reporter kinase in a sample, comprising: an elongate flow matrix, wherein said flow matrix comprises: (i) a sample-receiving zone; and (ii) a detection zone, located downstream of the sample-receiving zone, comprising a mixture of ADP and a bioluminescent reagent and (ill) a background-reduction zone, situated between the sample-receiving zone and the detection zone, that removes or inhibits non-reporter kinase present in the sample; wherein, in use, a sample is applied to the sample-receiving zone and migrates along the flow matrix through the background-reduction zone where non-reporter kinase is substantially removed or inhibited, and then into the detection zone where ATP generation is detected.
[19] 19. A lateral flow device for use in an assay for detecting the presence of an analyte in a sample, comprising: a backing strip on which is positioned an elongate flow matrix, wherein said flow matrix comprises: (i) a sample-receiving zone containing a reporter kinase attached to the flow matrix via a linker comprising a binding agent specific for the analyte; WO 2010/079357 PCT/GB2010/050018 104 (ii) a detection zone, located downstream of the sample-receiving zone. and containing a mixture of ADP and a bioluminescent reagent and (Wl) a background-reduction zone, situated between the sample-receiving zone and the detection zone, that removes or inhibits non-reporter kinase present in the sample; wherein, in use, a sample is applied to the sample-receiving zone and analyte present in the sample displaces reporter kinase from the flow matrix, said displaced reporter kinase migrates through the background-reduction zone where non-reporter kinase is substantially removed or inhibited, and then into the detection zone where ATP generation is detected.
[20] 20. A device according to Claim 18 or Claim 19, further comprising a background reduction zone, situated between the sample-receiving zone and the detection zone, wherein any ATP present is substantially removed.
[21] 21. A device according to any of Claims 1820, wherein the background-reduction zone comprises one or more of a substance that substantially inhibits or removes non reporter kinase and/ or ATP. such as an immobilised ATPase. an anionic or cationic exchange matrix, and/or a size exclusion matrix.
[22] 22. A device according to any of Claims 18-21 wherein the device is portable.
[23] 23. A method for detecting the activity of a reporter kinase in a sample, wherein the method is conducted using a device according to any of Claims 18 or 20-22, comprising: (i) applying the sample to the sample-receiving zone; (ii) allowing the sample to migrate to the detection zone; and (iii) detecting light output from the detection zone.
[24] 24. A method for detecting the presence of an analyte in a sample, wherein the method is conducted using a device according to any of Claims 19-22, comprising: (i) applying the sample to the sample-receiving zone; (ii) allowing any reporter kinase displaced from the sample-receiving zone to migrate to the detection zone; and WO 2010/079357 PCT/GB2010/050018 105 (iii) detecting light output from the detection zone.
[25] 25. A method according to Claim 23 or Clairn 24, wherein step (iii) is carried out by snapping off the detection zone of the device and placing it into a luminometer.
[26] 26. An assay according to any of Claims 1-9, or a method according to any of Clains 10-17 or 23-24, further comprising the step of recording the light output data obtained in step (ii) on a suitable data carrier.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US9416396B2|2016-08-16|Covalently linked thermostable kinase for decontamination process validation

---

US9046523B2|2015-06-02|Rapid bioluminescence detection system

---

US20090186368A1|2009-07-23|Assay with reduced background

---

Morlot et al.2013|Interaction of P enicillin‐B inding P rotein 2x and Ser/Thr protein kinase StkP, two key players in S treptococcus pneumoniae R 6 morphogenesis

---

US10466245B2|2019-11-05|Covalently linked thermostable kinase for decontamination process validation

---

US20070238145A1|2007-10-11|Phenotypic engineering of spores

---

US11085065B2|2021-08-10|Phenotypic engineering of spores

---

JP6082999B2|2017-02-22|Thermal stability assay reagent

---

CN102325897B|2016-12-14|Rapid bioluminescence detection system

---

JP2001204496A|2001-07-31|Method for assaying atp

---

JP2010537635A|2010-12-09|Analyte detection by coupled molecular binding enzyme assay

---

CA2849098A1|2013-04-11|Assay for clostridium botulinum neurotoxin

---

JP2003038161A|2003-02-12|Gene examination vessel

---

Murdoch2008|Development of a Novel Ultra-Sensitive Immunoassay for the Detection of Prion Protein on Surgical Instruments

同族专利:

---

公开号 | 公开日

---

US8512970B2|2013-08-20|

---

JP6042066B2|2016-12-14|

---

PL2385988T3|2016-11-30|

---

US20140030740A1|2014-01-30|

---

HK1158272A1|2012-07-13|

---

US9046523B2|2015-06-02|

---

EP2385988A1|2011-11-16|

---

GB0900151D0|2009-02-11|

---

CA2749021A1|2010-07-15|

---

JP2012514453A|2012-06-28|

---

ES2588181T3|2016-10-31|

---

WO2010079357A1|2010-07-15|

---

US20110318729A1|2011-12-29|

---

CN102325897A|2012-01-18|

---

CA2749021C|2018-06-12|

---

AU2010204173B2|2015-11-19|

---

ZA201104929B|2012-03-28|

---

EP2385988B1|2016-05-25|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

JP2856339B2|1991-02-21|1999-02-10|キッコーマン株式会社|Enzyme immunoassay|

---

GB9414096D0|1994-07-13|1994-08-31|Secr Defence|Labelled capture assay|

---

GB9902659D0|1999-02-05|1999-03-31|Microbiological Res Authority|Assay with reduced background|

---

GB9911095D0|1999-05-13|1999-07-14|Secr Defence|Microbiological test method and reagents|

---

GB2370113A|2000-12-15|2002-06-19|Lumitech|Method for determining cytolysis and cytotoxicity|

---

US6811990B1|2001-02-13|2004-11-02|Michael J. Corey|Methods and compositions for coupled luminescent assays|

---

GB0308199D0|2003-04-09|2003-05-14|Acolyte Biomedica Ltd|Combined microbiology system|

---

GB0406427D0|2004-03-22|2004-04-21|Health Prot Agency|Biological indicator|

---

US7355027B2|2004-06-16|2008-04-08|Dynport Vaccine Company Llc|Bacillus anthracis protective antigen|

---

GB0803068D0|2008-02-20|2008-03-26|Health Prot Agency|Cross-linked biological indicator|

---

GB0900151D0|2009-01-07|2009-02-11|Health Prot Agency|rapid bioluminescence detection system|GB0900151D0|2009-01-07|2009-02-11|Health Prot Agency|rapid bioluminescence detection system|

---

GB201115911D0|2011-09-14|2011-10-26|Health Prot Agency|Thermostable assay reagents|

---

US9562253B1|2012-11-09|2017-02-07|Point Of Care Diagnostics, Llc|Distinguishing between a bacterial and non-bacterial infection at the point of care|

---

DE102013205296A1|2013-03-26|2014-10-02|Olympus Winter & Ibe Gmbh|Method and system for monitoring a reprocessing device for endoscopes|

---

EP3902568A1|2018-12-28|2021-11-03|ASP Global Manufacturing GmbH|Article, system, and method for indication of treatment|

法律状态:
2013-08-29| PC1| Assignment before grant (sect. 113)|Owner name: THE SECRETARY OF STATE FOR HEALTH Free format text: FORMER APPLICANT(S): HEALTH PROTECTION AGENCY |

---

2016-03-17| FGA| Letters patent sealed or granted (standard patent)|

---

2021-09-09| PC| Assignment registered|Owner name: SECRETARY OF STATE FOR HEALTH AND SOCIAL CARE Free format text: FORMER OWNER(S): THE SECRETARY OF STATE FOR HEALTH |

优先权:

---

申请号 | 申请日 | 专利标题

---

GB0900151A|GB0900151D0|2009-01-07|2009-01-07|rapid bioluminescence detection system|

---

GB0900151.2||2009-01-07||

---

PCT/GB2010/050018|WO2010079357A1|2009-01-07|2010-01-07|Rapid bioluminescence detection system|

---