 Urate Oxidase
专利摘要:
The present invention relates to urate oxidase (uritase) proteins and nucleic acid molecules encoding the same. More specifically, the present invention relates to uricase proteins useful for preparing improved modified uricase proteins, for example with reduced immunogenicity and improved biocompatibility. 公开号:KR20010053633A 申请号:KR1020017001618 申请日:1999-08-05 公开日:2001-06-25 发明作者:마이클 허쉬필드；수잔 제이. 켈리 申请人:듀크 유니버시티； IPC主号:
专利说明:
Urate Oxidase Gout is the most common inflammatory joint disease found in people over age 40 (Roubenoff 1990). Painful gouty arthritis occurs when increased uric acid (hyperacidemia) in the blood causes the transient formation of microscopic crystals of monosodium urate monohydrate in the joint. Over time, chronic uric acid hyperemia also leads to destructive crystalline urate deposits (gout ash) in joints, soft tissues, and other tissues (Hershfield 1996). Uric acid has limited solubility in urine and, if overdose (hyperaciduria), causes kidney stone (urolithiasis). In malignant patients, especially in the case of leukemias and lymphomas, pronounced uric acid hyperemia and hyperuricuria (due to enhanced tumor cell replacement and destruction during chemotherapy) pose a serious risk of acute obstructive kidney disorder. (Sanberg et al. 1956; Gold and Fritz 1957; Cohen et al. 1980; Jones et al. 1990). Severe uric acid hyperemia and gout are accompanied by kidney abnormalities for a variety of causes, including cyclosporine treatment to inhibit tissue allograft rejection (West et al. 1987; Venkataseshan et al. 1990; Ahn et al. 1992; Delaney et al. 1992; George. And Mandell 1995). Hyperuricemia is caused by the overproduction and under-release of urate (Hershfield and Seegmiller 1976; Kelley et al. 1989; Becker and Roessler 1995). If uric acid hyperemia is mild, it can be controlled by food, but uric acid urinary agents that promote the release of uric acid (inefficient if the function of the kidney is impaired, if the symptoms are obvious and have serious clinical consequences). Or medication such as xanthine oxidase inhibitor allopurinol, which inhibits urate formation. Allopurinol is used to treat patients with nodular gout, kidney failure, leukemia and certain genetic disorders. Treatment for uric acid hyperemia is generally effective. However, in patients with malformed nodule gout, all conventional treatments are ineffective (Becker 1988; Fam 1990; Rosenthal and Ryan 1995). Moreover, ˜2% of patients treated with allopurinol develop allergic reactions and ˜0.4% develop severe hypersensitivity (Singer and Wallace 1986; Arellano and Sacristan 1993). The above life-threatening symptoms can cause acute renal failure, liver failure and severe skin damage (addictive epidermal lysis, deprived dermatitis, polymorphic erythema, Stevens Johnson syndrome). Allopurinol also inhibits the metabolism of azathioprine and 6-mercaptopurine, medications used to treat leukemia and inhibit tissue allograft rejection, resulting in significant uric acid hyperemia, which causes severe gout Or threaten kidney function. Ultimately, uric acid hyperemia is caused by mutations and inactivation of human genes for urate oxidase (ureate degrading enzymes) during the evolutionary process (Wu et al. 1989; Wu et al. 1992). Active urate degrading enzymes in hepatic peroxysomes in most primates and other mammals, except humans, convert urate to allantoin (+ CO 2 and H 2 O 2 ), which allotons are 80-100 higher than uric acid. It is twice as soluble and processed more efficiently by the kidneys. The parenteral uric acid degrading enzyme (Uricozyme ® , Clin-Midy, Paris) from Aspergillus flavus is used to treat severe uric acid hyperemia associated with leukemia chemotherapy for more than 20 years in France and Italy. (London and Hudson 1957; Kissel et al. 1968; Brogard et al. 1972; Kissel et al. 1972; Potaux et al. 1975; Zittoun et al. 1976; Brogard et al. 1978; Masera et al. 1982) and used in recent clinical trials of leukemia patients in the United States (Pui Et al. 1997). Uricase is a faster onset of action than allopurinol (Masera et al. 1982; Pui et al. 1997). In patients with gout, the infusion of uric acid enzymes suppresses acute attacks and reduces the size of gout (Kissel et al. 1968; Potaux et al. 1975; Brogard et al. 1978). Although daily injection of A. flavus urate dehydrogenase is an effective method of treating acute uric acid hyperemia during the short term of chemotherapy, there are serious drawbacks in treating gout of recurrence or nodule of nodules. In addition, the efficacy of A. flavus urease is dramatically reduced in patients producing anti-ureta lyase enzymes (Kissel et al. 1968; Brogard et al. 1978; Escudier et al. 1984; Mourad et al. 1984; Sibony et al. 1984). Serious allergic reactions occur, including anaphylaxis (Donadio et al. 1981; Montagnac and Schillinger 1990; Pui et al. 1997). Long-term acting and reduced immunogenicity, uric acidase is urgently needed for chronic treatment. One approach to sequestering adventitious enzymes from proteases and the immune system involves a method of covalently binding monomethoxypolyethylene glycol (PEG), an inert, non-toxic polymer, to the surface of a protein (Harris and Zalipsky 1997). In the early days, the use of PEGs with Mr ˜1,000 to> 10,000 has been reported to extend the circulating life and reduce immunogenicity of many foreign proteins in animals (Abuchwski et al. 1977a; Abuchowski et al. 1977b; Davis et al. 1981a; Abuchowski et al. 1984; Davis et al. 1991). In 1990, bovine adenosine deaminase (ADA) (PEG-ADA, ADAGEN ® produced by Enzon), modified with PEG of Mr 5000, was first recognized by the US Food and Drug Administration for severe combined immunodeficiency due to ADA deficiency. PEGylation protein used for the treatment of (Hershfield et al. 1987). It has been shown in the past 12 years that during the long-term treatment with PEG-ADA, anti-ADA antibodies can be detected by sensitive ELISA in most patients, but no allergic or sensitive reactions occur; PEG-ADA was quickly eliminated in some patients producing anti-ADA antibodies, but this was usually transient (Chaffee et al. 1992; Hershfield 1997). It should be recognized that during treatment with PEG-ADA, it is common for immune function in patients with ADA deficiency to be normal (Hershfield 1995; Hershfield and Mitchell 1995). Therefore, immunogenicity appears to be more important when developing PEGylation enzymes for long-term treatment of patients with normal immune function. Immunogenicity is associated with the induction of immune responses by injected antigen preparations (such as PEG-modified proteins or unmodified proteins), while antigenicity is known to those skilled in the art to indicate the reaction of antigens with existing antibodies. Overall, antigenicity and immunogenicity represent immune responses. In previous studies of PEG-ureatase, immune responses were analyzed in a variety of ways: in vitro reactions of PEG-urease with antibodies; Measurement of induced antibody synthesis; And increased removal rate after repeated injections. PEGylation has been reported to reduce the immunogenicity and prolong cycle life of fungi and swine urate dehydrogenase in animals (Chen et al. 1981; Savoca et al. 1984; Tsuji et al. 1985; Veronese et al. 1997). PEG-modified Candida uratease rapidly degraded serum urate in five human volunteers of normal uric acid to undetectable levels (Davis et al. 1981b). PEGylated Arthrobacter produced by Enzon has been used in the treatment of patients with allopurinol-hypersensitive leukemia and renal failure and significant uric acid hyperemia (Chua et al. 1988; Greenberg and Hershfield 1989). Four intramuscular injections were administered over about 2 weeks. During this short period of time, uric acid hyperemia was controlled and no anti-ureatase antibody was detected by ELISA in the patient's plasma. No further use and clinical trials of the medicament have been made. To date, for the stability and reliable use in long-term treatments, no development of uricase or PEG-ureatase with suitable long-term circulating life and sufficiently reduced immunogenicity has been made. It is an object of the present invention to provide improved forms of urate and PEGylated urateases that can meet the above needs. The present invention is a unique recombinant uric lyase in mammals, which is mutated in a way that improves the PEGylation ability to strongly mask immunogenic epitopes. This application claims the benefit of US Provisional Patent Application No. 60 / 095,489, filed August 6, 1998, the entire contents of which are incorporated herein by reference. The invention disclosed herein is made with the support of the United States Government under Grant No. DK48529, issued by the National Institutes of Health. The government has rights in the invention. The present invention relates generally to urate oxidase (ureatase) proteins and nucleic acid molecules encoding the same. More specifically, the present invention relates to uricase, which is particularly useful as an intermediate suitable for preparing improved modified uricase protein with reduced immunogenicity and increased biocompatibility. Preferred modified uricase proteins of the present invention include uric acid enzymes covalently linked to poly (ethylene glycol) or poly (ethylene oxide). Accordingly, the present invention relates to a uratase protein, an antibody that specifically binds to the protein, a nucleic acid molecule encoding the urate enzyme protein and useful fragments thereof, a vector comprising the nucleic acid molecule, a host cell comprising the vector And it provides a method and a method of using the uricase protein and the nucleic acid molecule. 1 shows SDS-mercaptoethanol PAGE (12% gel) analysis, Figure 2 shows the circulating life of natural and PEGylated PBC urease. 3 is a graph showing the relationship between the concentrations of serum urinase and uric acid in serum and urine; 4 shows the maintenance of the circulating amount of uric acid enzyme activity (measured in serum) after repeated injections, Fig. 5 shows the amino acid sequence inferred by comparing pig-bibi chimeric urease (PBC urease) and porcine urease (PKS urease) comprising the mutations R291K and T301S with those of pigs and baboons, Figure 6 is a view comparing the amino acid sequence of PKS and pig uric acid enzyme, 7 is a view comparing amino acid sequences of PBC and PKS, 8 is a view comparing the amino acid sequence of PBC and urease of pigs, 9 is a view comparing the amino acid sequence of porcine uric acid enzyme and D3H, 10 is a view comparing amino acid sequences of PBC and D3H, 11a and 11b show the cDNA coding sequence of PKS and porcine urate lyase in Best Fit (GCG software), 12A and 12B show the cDNA coding sequence of PKS and BB uratease in Best Fit (GCG software). 13a and 13b are comparisons of cDNA coding sequences of PBC and porcine urate lyase in Best Fit (GCG software), 14A and 14B show the best-fit (GCG software) comparison of cDNA coding sequences of PBC and BB uratease; It is a primary object of the present invention to provide novel uric acid enzyme proteins and nucleic acid sequences encoding the same. Another object of the present invention is to provide a method for purifying uric acid enzyme protein prepared by a recombinant method as disclosed herein. Still another object of the present invention is to provide a method for reducing the amount of uric acid in a body fluid of a mammal by administering to the mammal a composition comprising the uricase protein of the present invention. Another object of the present invention is to provide an antibody against the uricase protein disclosed herein. It is another object of the present invention to provide a vector and host cell comprising a nucleic acid sequence disclosed herein and a method for producing a uratease protein encoded by the sequence. The present invention provides a urate dehydrogenase protein that is used to prepare PEG-ureatase that is substantially non-immunogenic while retaining all or almost all of the unmodified urate degrading activity. Uric acid degrading activity is expressed herein as international units (IU) per mg protein, and the IU of urate degrading activity is defined as the amount of enzyme that consumes 1 μmole of uric acid per minute. The present invention provides a mammalian recombinant uricase protein that has been modified to insert one or more lysine residues. As used herein, recombinant protein refers to any protein produced artificially and is to be distinguished from a naturally produced protein (ie, produced in the tissue of an animal containing only the natural gene for the particular protein desired. ). Proteins include peptide and amino acid sequences. The recombinant uricase protein of the present invention may be chimeric or hybrid of two or more mammalian protein, peptide or amino acid sequences. According to a preferred embodiment of the present invention, the present invention can be used to prepare a recombinant uric acidase protein of a mammal, and is mutated to increase the number of lysine at the position where the protein is PEGylated, the PEGylated Uricase is substantially the same enzyme activity as unmodified uricase, and PEGylated uricase shows acceptable immunogenicity. Also included in the present invention are truncated forms without the amino and / or carboxyl termini of the urate degrading enzymes of the present invention. Preferably, the urate dehydrogenase of the present invention is in a form that is not cleaved enough to remove lysine. Those skilled in the art will recognize that the conjugated urease-carrier complex should not contain as many bonds as to substantially reduce the activity of urease or contain too few bonds to retain unacceptable immunogenicity. Doing. Preferably, the conjugate retains at least about 70% to about 90% of the uric acid degrading activity of the unmodified urate dehydrogenase protein, maintains enzymatic activity during storage, and maintains the mammalian plasma and / or at physiological temperature. It is more stable compared to uricase protein, which is not modified enough to maintain enzyme activity in serum. At least about 80% to about 85% retention of urea degradation activity is acceptable. In addition, in a preferred embodiment of the present invention, the conjugate exhibits substantially reduced immunogenicity and / or immune response compared to unmodified uratease protein. In an embodiment of the present invention, the present invention may be modified by binding a non-toxic, non-immunogenic, pharmaceutically acceptable carrier such as PEG by covalent linkage to at least one lysine in the uricase protein. It provides a uric acid enzyme protein disclosed in. In another variation of the invention, the uricase protein is modified by covalent linkage with a carrier through up to about 10 lysines in its amino acid sequence. Binding via any of lysine 2, 3, 4, 5, 6, 7, 8 or 9 is also included in another variant of the invention. The urease protein of the present invention is a recombinant molecule comprising a fragment of the urease protein of the liver of pigs and baboons. Modified baboon sequences are also provided. In an embodiment of the present invention, the present invention provides chimeric swine-bi-biurease (PBC urease (SEQ ID NO: 2)), wherein the enzyme comprises amino acid sequence 1-225 (SEQ ID NO: 7) of swine urate dehydrogenase and The amino acid sequence 226-304 (SEQ ID NO: 6) of the urolytic enzyme (as shown in Figure 5). In another embodiment of the present invention, the present invention provides chimeric swine bead urease (PKS urease), wherein the enzyme is amino acid sequence 1-288 of urease of pig and amino acid of urease of bab SEQ ID NOs: 289-304 (SEQ ID NO: 4). Truncated forms of PBC and PKS also belong to the present invention. Preferred cleavage forms are truncated PBCs and PKS that have been eliminated six amino termini or three carboxyl termini, or both. Representative sequences are described in SEQ ID NO: 8 (PBC amino terminal truncation), SEQ ID NO: 9 (PBC carboxyl terminal truncation), SEQ ID NO: 10 (PKS amino terminal truncation) and SEQ ID NO: 11 (PKS carboxyl terminal truncation). Each of PBC urease, PKS urease, and their cleavage forms, has one to four more lysines than those found in other mammalian urease. The present invention provides nucleic acid (DNA and RNA) molecules (sequences), including the isolated, purified and / or cloned forms of the nucleic acid molecules encoding the uricase protein and truncated forms disclosed herein. Preferred embodiments are shown in SEQ ID NO: 1 (PBC urate lyase) and SEQ ID NO: 3 (PKS urate lyase). A vector comprising the nucleic acid molecule described above (for expression and cloning) or provided by the present invention. The present invention also provides a host cell comprising the vector described above. Also provided is an antibody that specifically binds to the urate enzyme protein of the invention. Antibodies to the amino terminal portion of swine uratease and antibodies to the carboxyl terminal portion of the urine urease should be useful for detecting PBC, or other similar chimeric proteins, when used in conjunction. Preferably, the antibody to the amino terminal portion of the chimeric uricase enzyme should not recognize the amino terminal portion of the ubiquinate urate enzyme, and similarly the antibody to the carboxyl terminal portion of the chimeric uricase enzyme is the The carboxyl terminal portion of the degrading enzyme should not be recognized. More preferably, antibodies are provided that specifically bind to PBC or PKS but do not bind to native proteins such as swine and / or baboons. In another embodiment of the invention, the invention is used in the manufacture of a pharmaceutical composition capable of reducing the amount of uric acid in body fluids, such as urine and / or serum or plasma, wherein the composition is a urease protein disclosed herein. Or at least one of the uricase enzyme conjugates and a pharmaceutically acceptable carrier, diluent or excipient. The invention is also used in a method of reducing the amount of uric acid in mammalian body fluids. The method provides a mammal with a uric acid-lowering effective amount of a composition comprising a urate dehydrogenase protein or urate degrading enzyme conjugate and a diluent, carrier or excipient, preferably a pharmaceutically acceptable carrier, diluent or excipient. Administering. The mammal to be treated is preferably a human. Said administration step may preferably be carried out by intravenous, subcutaneous, transdermal, intramuscular or intranasal injection. Elevated uric acid amounts may be in the blood or urine and may be associated with gout, gout, kidney failure, transplantation or malignant disease. In another embodiment of the present invention, the present invention provides a method for isolating and purifying urate lyase from, for example, a urease solution containing, for example, cellular and subcellular fragments made from recombinant manufacturing processes. Preferably, the purification method utilizes a limited solubility of mammalian urate dehydrogenase (Conley et al. 1979) at low pH, which washes the crude recombinant extract at a pH of about 7 to about 8.5, mostly dissolved in a low pH range. Protein is removed, and then active uricase is dissolved in a buffer, preferably sodium carbonate buffer, at pH about 10-11, preferably about 10.2. The dissolved active uricase is then applied to an anion exchange column, such as a Q Sepharose column washed with a salt concentration gradient in buffer at pH about 8.5, and then sodium carbonate at pH about 10 to about 11, preferably about 10.2. Elution with a gradient of sodium chloride in the buffer yields purified uric acid enzyme. The enzyme may additionally be purified by gel filtration chromatography at pH about 10 to about 11. The enzyme can be further purified by reducing the pH to about 8.5 or less so as to selectively precipitate uricase but not to precipitate more soluble contaminants. After washing at low pH (7-8), the urease is solubilized at pH about 10.2. Uricase enzyme products can then be analyzed by methods known in the pharmaceutical arts, such as high performance liquid phase chromatography (HPLC), other chromatography methods, light scattering, centrifugation and / or gel electrophoresis. The present invention provides urate lyase enzymes, which are useful intermediates for the preparation of improved urate lyase enzyme conjugates with water-soluble polymers, preferably poly (ethylene glycol) or poly (ethylene oxide) and urate lyase. Urinalytic enzymes herein include, unless otherwise indicated, natural tetramers as well as individual subunits. Although humans do not produce active urate enzymes, urate enzyme transcripts are amplified from human liver RNA (Wu et al. 1992). It is theoretically possible for any human urate transcript to be translated; Even if the peptide product is not full length or unstable, the product can be processed by the antigen presenting cells and can serve to determine the immune response to foreign uric acid enzymes used in therapy. Theoretically, it is possible to reconstruct and express human urate dehydrogenase cDNA by eliminating two known nonsense mutations. However, after the introduction of the first nonsense mutation, in the absence of selection pressure, it is easy to accumulate nonsense mutations that are harmful to human genes for millions of years (Wu et al. 1989; Wu et al. 1992). It is very difficult to identify and “correct” all the variants that can achieve optimal enzyme activity and protein stability. We found that there is a high homology (similarity) between the amino acid sequence of human uric acid enzyme inferred against pig uric acid enzyme (approximately 86%) and the amino acid sequence of BB uric acid enzyme (about 92%). 6-14, Example of similarity measurement, while there is <40% homology (similarity) between human and A. flavus uratease (Lee et al. 1988; Reddy et al. 1988; Wu et al. 1989; Legoux et al. 1992; Wu et al. 1992). The present invention provides chimeric urease proteins produced recombinantly from two different mammals, which proteins are designed to reduce immunoreactivity in humans compared to fungal and bacterial enzymes that are clearly involved. The use of urate dehydrogenase derivatives in mammals is expected to be more acceptable to patients and physicians. Experiments have demonstrated that active PEGs used to prepare PEG-ADA and modify other proteins are bound through the primary amino group of the amino terminal residue (when the amino group is present and not blocked) and the epsilon-amino group of lysine. . The above strategy is useful because relaxed reaction conditions can be used and lysine charged with cation tends to be located on the surface of the protein. The desired effect of PEGylation may be due in part to the properties of the PEG polymer (eg, mass, branched or unbranched structure, etc.) as well as structural factors that determine the function and removal rate of the protein and PEG binding of the protein to the epitope. The latter of these reasons is important because it also depends on the relative number and distribution of sites. Strategies to improve PEGylation capacity to mask epitopes and reduce immunogenicity by way of semi-white introduction of new lysine residues for PEG addition have been studied (Hershfield et al. 1991). The strategy above employs mutagenesis to replace selected arginine codons with lysine codons, which substitutions maintain positive charges and have minimal impact on computer-predictive indicators of surface likelihood and antigenicity (when amino acid sequences are made) Only useful). As an experimental test of the above strategy, recombinant E. coli purine nucleoside phosphorylase (ENEP) (Hershfield et al. 1991) was used. Substitution from Arg to Lys at three locations was introduced, the increased number of lysine per subunit was 14-17, and the catalytic activity was unchanged. Purified triple-variants retained full activity even after modifying ˜70% of the accessible NH 2 groups with excess disuccisinyl-PEG5000. It was found by titration of reactive amino groups before and after PEGylation that the triple variant can accommodate one more PEG per subunit than the wild type enzyme. PEGylation increased the cycle life of wild-type and variant EPNP enzymes in mice from ˜4 hours to> 6 days. After a series of weekly / weekly intraperitoneal injections, in all mice treated with two unmodified EPNPs and 10 of 16 mice injected with PEGylated wild-type EPNP, high concentrations and circulating lifespan of anti-EPNP antibodies were observed. A significant decrease in was observed. In contrast, only 2 of 12 mice treated with variant PEG-EPNP showed rapid clearance; Low concentrations of antibody in these mice were independent of circulating life. Thus, the strategy described above was successful in substantially reducing immunogenicity although only one of the three lysines was modified after treatment with active PEG. Each of the baboon and porcine urate dehydrogenase subunits consists of 304 amino acid sequences, of which 29 (ie, 1 per about 10 residues) are lysine. Attempts to substitute Lys for 2 Arg in cloned cDNA for ubiquinate uratease, and Glu codon-lysed Lys at position 208, known as Lys in the human uratease gene, significantly reduced uricase activity. Resulting in the expressed variant protein of the baboon. After the mutation of arginine to lysine in the mammalian DNA sequence occurred, it became clear from the above experiment that the ability to maintain the activity of uratease was unpredictable. Subsequently, it became evident that amino acid residue 291 in BB uricase was lysine and the residue in swine was arginine. The ApaI restriction site present in both cDNAs was used to construct chimeric urateases, where the first 225 amino acids in the chimeric protein were derived from porcine cDNA, and the 79 carboxyl ends were derived from baboon cDNA. will be. The resulting porcine-bi-chimeric (PBC) uratease (SEQ ID NO: 2) has 30 lysines and one more lysine than the “parental” enzyme. An additional feature of PBC urease is that its "bibe" portion differs from four out of 79 amino acid residues compared to human urease, while pig and human urease differ ten from the same site. A modified form of PBC was then constructed, which retained the extra lysine at position 291, differing from porcine urate lyase by replacing threonine with serine at residue 301 only (“pig KS” urate lyase). (SEQ ID NO: 4)). In view of the above results, where several lysine insertions have a deleterious effect on activity, whether PBC and PKS chimeric urease show the same complete activity as compared to unmodified native porcine urease and not mutated It was unpredictable whether it exhibited four times more activity than the natural non- urinary lyase. The present invention provides a recombinant swine-bi-bee chimeric urease comprising a portion of the liver and baboon's hepatic urease sequence. An example of such chimeric urate enzymes includes the first 225 amino acids (SEQ ID NO: 7) derived from the swine urate enzyme sequence (SEQ ID NO: 7) and the latter 79 amino acids (SEQ ID NO: 6) derived from the ubiquinate urate enzyme sequence (Swine-BB Uricase, or PBC Uricase; FIG. 6 and SEQ ID NO: 2). Other examples of chimeric urate enzymes of the invention include the first 288 amino acids (SEQ ID NO: 7) derived from the swine urate enzyme sequence (SEQ ID NO: 7) and the latter 16 amino acids (SEQ ID NO: 6) derived from the baboon urate enzyme sequence do. Since the latter sequence differs from the pig's sequence at only two positions, ie it has lysine (K) instead of arginine at residue 291 and serine (S) instead of threonine at residue 301, the variant is swine-KS. Or PKS uric acid dehydrogenase. A vector (for expression and cloning) is provided comprising a nucleic acid molecule encoding a protein of the invention. Preferred vectors include those exemplified herein. It will be apparent to those skilled in the art that the nucleic acid molecule can be inserted into an expression vector, such as a plasmid, in a suitable directional and accurate reading frame for expression. Regulatory sequences are generally present in the expression vectors employed and are known in the art, but where necessary, the nucleic acid (DNA) can be linked to suitable transcriptional regulatory and translational regulatory sequences recognized by the host. The vector of the present invention is then introduced into the host cell using standard techniques. In general, not all host cells are transformed with the vector. Thus, there is a need for a process for selecting transformed host cells. A selection method known in the art is to insert a DNA sequence, such as an antibiotic resistance gene, that encodes a selective marker property in a transformed cell into an expression vector with the necessary regulatory sequences. As a variant of the invention, the gene encoding the above-described selective properties may be in another vector used to co-transform the host cell. Vectors of the invention may also include suitable promoters, and may also include prokaryotic promoters that allow for expression (transcription and translation) of DNA in bacterial host cells such as E. coli. Many expression systems are known and known in the art and include bacteria (such as E. coli and Bacillus subtilis), yeast (such as Saccharomyces cerevisiae), filament fungi (such as Aspergillus), plant cells , Animal cells and insect cells. Suitable vectors may include, for example, prokaryotic repricons such as ColE1 ori for proliferation in prokaryotic cells. Typical prokaryotic vector plasmids are pUC18, pUC19, pUC322 and PBR329 available from Biorad Laboratories (Richmond, CA) and pTcr99A and PKK223-3 available from Pharmacia (Piscataway, NJ). The vector utilizes an SV40 late promoter that promotes expression of the cloned gene and promotes the highest expression in T antigen-producing cells such as COS-1 cells. An example of an expression vector of an inducible mammal is pMSG, available from Pharmacia. The vector uses a glucocorticoid-induced promoter of long terminal repeats of mouse mammalian tumor virus to promote expression of the cloned gene. Useful yeast plasmids are pRS403-406 and pRS413-416, which are available from Stratagene Cloning System (LaJolla, Calif.). Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast insertion plasmids (Yips) and insert yeast selective markers HIS3, TRP1, LEU2 and URA3. Plasmid pRS413-416 is a yeast centrifuge plasmid (Ycps). The present invention also provides a host cell comprising the vector described above. Preferred host cells include those exemplified and described herein. Uricase enzymes of the invention can be conjugated to relatively few PEG strands through biologically stable, nontoxic covalent bonds, which increase the biological half life and solubility and reduce immunogenicity of the enzyme. Such bonds include urethane (carbamate) bonds, secondary amine bonds and amide bonds. Various active PEGs suitable for such conjugation are available from Shearwater Polymers, Huntsville, AL. The invention can also be used to prepare pharmaceutical compositions of uricase protein as conjugates. The conjugates are substantially immunogenic and have at least 70%, preferably 80%, more preferably about 90% or more of the uric acid degrading activity of the unmodified enzyme. Water soluble polymers suitable for use in the present invention include those referred to collectively as linear and branched poly (ethylene glycol) or poly (ethylene oxide), PEGs. An example of a branched PEG is the subject of US Pat. No. 5,643,575. According to an embodiment of the invention, the average number of lysines inserted per uricase subunit is 1-10. In a preferred embodiment of the present invention, the number of lysine added per urate dehydrogenase subunit is 2-8. Obviously, the number of lysines added should be such that they do not adversely affect the activity of uric acid degrading enzymes. The PEG molecules of the conjugates are preferably conjugated via lysine of the urate dehydrogenase protein, more preferably via a lysine inserted into a protein that does not originally have a lysine at a non-natural lysine or insertion position. The present invention provides a method of increasing a useful harmless PEG binding site, the method comprising the step of mutating a native uricase protein to insert at least one lysine residue. Preferably, the method is to substitute arginine with lysine. The PEG-ureatase conjugates prepared by the present invention are useful for reducing the concentration of uric acid (ie, reducing the amount) of uric acid in the blood and / or urine of mammals, preferably humans, and include gout, gout, kidney failure, It can be used to treat increased amounts of uric acid associated with tissue transplantation and malignant disease. PEG-urease conjugates can be introduced into mammals with excessive uric acid concentrations through a variety of routes, which can be administered by oral, enema or suppository, intravenous, transdermal, subcutaneous, intramuscular and intraperitoneal routes. . Patton, JS et al., (1992) Adv Drug Delivery Rev 8: 179-228. Effective dosages of PEG-urease are determined by the uric acid concentration and size of the individual. In an embodiment of the invention, the PEG-urease is dissolved and administered in a pharmaceutically acceptable excipient or diluent within the range of 10 μg to about 1 g. In a preferred embodiment of the invention, the dosage is about 100 μg to 500 mg. More preferably, the conjugate uratease is administered at 1 mg to 100 mg, such as 5 mg, 20 mg or 50 mg. Mass at the dosage of an embodiment of the invention refers to the amount of protein in the conjugate. Pharmaceutical formulations comprising PEG-urease may be prepared according to conventional techniques such as those disclosed by Remington's Pharmaceutical Sciences, (1985) Easton, PA: Mack Publishing. Excipients suitable for the preparation of injectable solutions include, for example, phosphate buffered saline, lactate Ringer's solution, water, polyols and glycols. Pharmaceutical compositions suitable for parenteral injection include pharmaceutically acceptable sterile water-soluble or non-aqueous liquids, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injections or dispersions immediately before use. Such formulations may include additional ingredients such as preservatives, solubilizers, stabilizers, wetting agents, emulsions, buffers, antioxidants and diluents. PEG-urease may also be provided as a sustained release composition for use in implantation into a subject, which will continue to regulate the increased amount of uric acid in the blood and urine. For example, polylactic acid, polyglycolic acid, regenerated collagen, poly L-lysine, sodium alginate, gellan gum, chitosan, agarose, multilayer liposomes and other depot formulations may be bioerodible which can be formulated with biologically active ingredients. Or a biodegradable material. The material, when implanted or injected, gradually releases the active material into surrounding tissue. For example, the method of enclosing PEG-ureatase is the method disclosed in US Pat. No. 5,653,974, which is incorporated herein by reference. The use of bioerodible, biodegradable and other depot agents is clearly contemplated in the present invention. The use of infusion pumps and matrix capture systems for the delivery of PEG-urease is also within the scope of the present invention. PEG-ureases may also be included preferably in micelles or liposomes. Liposomal enveloping techniques are known in the art. As a reference, Lasic, D et al., (Eds.) (1995) Stealth Liposomes, Boca Raton, FL: CRC Press. The PEG-ureatase pharmaceutical compositions disclosed herein are hemodialysis in patients at risk of urate-induced renal failure, such as tissue transplant recipients (Venkataseshan, VS et al. (1990) Nephron 56: 317-321) and malignant diseases. Reduce the need for In patients with large accumulations of crystalline urate (gout), the pharmaceutical composition improves the quality of life more quickly than currently available treatments. The following examples are intended to illustrate various aspects of the invention disclosed above and are not intended to limit the scope of the invention. Example 1 A. Construction of PBC, PKS and Related Uricase CDNAs Standard methods, and instructions supplied by reagent manufacturers for PCR amplification of urate oxidase cDNA (US Pat. Nos. 4,683,195 and 4,683,202, 4,965,188 & 5,075,216), and for cloning and cDNA Was used to obtain total RNA of cells for sequencing (Erich 1989; Sambrook et al. 1989; Ausubel 1998). PCR primers used in porcine and baboon urate oxidase (Table 1) were designed using published coding sequences (Wu et al. 1989) and PRIME software program (Genetics Computer Group). Porcine Liver Uricase cDNA: Sense: 5 'gcgcgaattccATGGCTCATTACCGTAATGACTACA 3' Antisense: 5 'gcgctctagaagcttccatggTCACAGCCTTGAAGTCAGC 3' D3H BB Liver Uricase CDNA: Sense: 5 'gcgcgaattccATGGCCCACTACCATAACAACTAT 3' Antisense: 5 'gcgcccatggtctagaTCACAGTCTTGAAGACAACTTCCT 3' Restriction sequences (lower case) introduced at the ends of the primers are sense (pig and baboon) for EcoRI and NcoI; Antisense (pig) is for NcoI, HindIII, XbaI; Antisense (BI) is for NcoI. In the case of BB's sense primers, the third codon GAC (asphatate) present in BB's urate oxidase (Wu et al. 1992) was replaced with CAC (histidine) and this codon was a human urate oxidase pseudo It exists at a corresponding position in the coding sequence of a psedogene (Wu et al. 1992). For this reason, the nasal recombinant urate oxidase produced from the use of the primers described above was named D3H nabiureate oxidase. Total RNA of cells from pig liver and baboon liver was reverse transcribed using a primary strand kit (Pharmacia Biotech, Piscataway, NJ). PCR amplification using Taq DNA polymerase (GibcoBRL, Life Technologies, Gaithersburg, Md.) Was performed using a program [30 s, 95 ° C .; 30 s, 55 ° C .; 60 cycles in a thermal cycler (Ericomp, San Diego, Calif.) Having 60 s, 70 ° C.], followed by [30 s, 95 ° C .; 60 s, 70 ° C.] was carried out for 10 cycles. Urate oxidase PCR products were digested with EcoRI and HindIII and cloned into pUC18 (pig) and also directly cloned using the TA cloning system (Invitrogen, Carlsbad, Calif.) (Pork and D3H babies). cDNA clones were transformed into E. coli XL1-Blue (Stratagene, La Jolla, Calif.). Plasmid DNA containing cloned urate lyase cDNA was prepared and the cDNA insertion sequence was analyzed by standard deoxygen technology. Clones with known urate oxidase DNA coding sequences (except for the D3H substitutions in the ratios of urate oxidases listed in Table 1) were constructed and demonstrated in serial experiments by standard recombinant DNA methods. Pig and D3H baboons containing full-length coding sequences were introduced into pET expression vectors (Novagen, Madison, WI) as follows. TA plasmids were cleaved with NcoI and BamHI restriction enzymes to obtain D3H biureta urate dehydrogenase cDNA followed by subcloning into NcoI and BamHI cloning sites of expression plasmids pET3d and pET9d. pUC plasmid clones were digested with EcoRI and HindIII restriction enzymes to obtain full-length porcine urate lyase cDNA, which was subcloned into the EcoRI and HindIII restriction sites of pET28b. Pig cDNA coding sites were also obtained by cleaving pET28b with NcoI and BlpI and introducing it into the NcoI and BlpI sites of the expression plasmid pET9d. The clone of pET3d-D3H-bibe was treated with NcoI-ApaI restriction enzyme to obtain a 624 bp fragment of D3Hbeo urinase cDNA, and the fragment of the D3Hbebe was a 624 bp NcoI-ApaI restriction enzyme fragment of the corresponding pig cDNA. And pig-bibi chimera (PBC) cDNA was constructed. The resulting PBC urate oxidase cDNA is composed of porcine urate oxidase codon 1-225 bound to the reading frame to codon 226-304 of the biferate urate oxidase. The clone of pET3d-D3H-bebe was treated with NcoI-NdeI restriction enzyme to obtain a 864 bp fragment of D3H bee urease cDNA, and the fragment of the D3H bee was 864 bp NcoI-NdeI restriction enzyme fragment of the corresponding pig cDNA. The porcine-KS urate oxidase (pig KS) cDNA was constructed by substituting. The resulting PKS urate oxidase cDNA consists of porcine urate oxidase codon 1-288 bound to the reading frame to codons 289-304 of baboon urate oxidase. The amino acid sequences of D3H baboon, swine, PBC and PKS urate oxidase are shown in FIG. 5 and in the Sequence Listing. A 15% glycerol scoop of each of the transformants was prepared using standard techniques and stored at -70 ° C. Each of the transformants was expressed and recombinant enzymes were isolated (Table 2). Pigs, PBC chimeras, and swine KS urease showed very similar intrinsic activity, which was higher than the intrinsic activity of BB recombinant uricase. It is about 4-5 times higher. The above order was confirmed through a number of different experiments. The intrinsic activity of PBC uricase obtained by a number of different methods varied in the 2-2.5 fold range. Comparison of Expressed Mammalian Recombinant Uricase ConstructSpecific activity * (unit / mg)Relative activity (chimera = 1) PBC7.021.00 Pig KS7.171.02 pig5.570.79 baboon1.360.19* The amount of protein was determined by Lowry method. Uricase activity was determined by spectrometer (Priest and Pitts 1972). The activity measurement was carried out at 23-25 ° C. in a quartz cuvette containing 1 ml reaction mixture (0.1 M sodium borate, pH 8.6, 0.1 mM uric acid). The loss of uric acid was measured by the decrease in absorbance at 292 nm. One international unit (IU) of uricase is an amount that catalyzes the loss of 1 μmol of uric acid per minute. E. coli BL21 (DE3) pLysS transformants of the four urate degrading enzyme cDNA-pET constructs described in Table 2 were directed to carbenicillin for the selective antibiotic (pET3d (Swine KS), as indicated in the pET system manual. And chloramphenicol); In the case of pET9d (PBC, pig, BB), it was placed in LB agar containing kanamycin and chloramphenicol. 5 ml cultures (LB and antibiotics) were inoculated with the transformed colonies alone and grown at 37 ° C. for 3 hours. A portion of 0.1 ml was then transferred to 100 ml of LB medium containing selective antibiotic and 0.1% lactose (for inducing the expression of uratease). Incubated overnight at 37 ° C., and bacterial cells from 0.5 ml of culture were extracted in SDS-PAGE loading buffer and analyzed by SDS-mercaptoethanol PAGE; This confirmed the expression of uricase protein in each of the four cultures (results not shown). Residual cells from each 100 ml culture were collected by centrifugation and washed with PBS. The cells are then resuspended in phosphate-buffered saline, pH 7.4 (PBS) containing 1 mM AEBSF protease inhibitor (Calbiochem, San Diego, Calif.) And digested on ice in a bacterial cell mill (Microfluidics, Boston, Mass.) It was. Insoluble material (including uric acidase) was precipitated by centrifugation (20,190 × g, 4 ° C., 15 minutes). The precipitate was washed twice with 10 mL of PBS and extracted overnight at 4 ° C. with 2 mL of 1 M Na 2 CO 3 , pH 10.2. The extract was diluted with 10 mL of water and centrifuged (20,190 × g, 4 ° C., 15 min). Uricase activity and protein concentration were determined. Example 2 Expression and Isolation of Recombinant PBC Uricase (Preparation in a 4 Liter Fermenter) According to the Novagen pET system manual, the pET3d-PBC urate dehydrogenase transformants were run from glycerol swabs onto LB agar plates containing carbenicillin and chloramphenicol. 200 ml inoculum starting from colonies alone was prepared at 37 ° C. with LB-antibiotic liquid medium on a rotary shaker (250 rpm) using the method described in the pET system manual to maximize pET plasmid retention. When OD 525 reached 2.4, cells were collected from the 20 ml culture by centrifugation and resuspended in 50 ml of fresh medium. The suspension was transferred to a high density fermentor containing 4 liters of carbenicillin and chloramphenicol-containing SLBH medium (the composition of the SLBH medium, the design and operation of the fermentor is described in Sadler et al. 1974). After 20 hours of growth in the presence of 32 ° C. and O 2 (OD 525 = 19), 0.4 mM of isopropylthiogalactoside (IPTG) was added to induce the production of urate. After an additional 6 hours (OD 525 = 19), bacterial cells were collected by centrifugation (10,410 xg, 10 minutes, 4 ° C), washed once with PBS and stored frozen at -20 ° C. Bacterial cells (189 g) were resuspended in 200 ml PBS and digested by refrigeration in ice / salt baths by sonication (Heat System Sonicator XL, probe model CL, Farmingdale, NY), 100% strength upon sonication. Burst 4 x 40 seconds and gave a minute break between bursts. PBS-insoluble material (including uricase) was precipitated by centrifugation (10,410 × g, 10 minutes, 4 ° C.) and washed five times with 200 ml PBS. Uricase in PBS-insoluble precipitate was extracted with 80 ml of 1 M Na 2 CO 3 , pH 10.2 containing 1 mM phenylmethylsulfonylfluoride (PMSF) and 130 μg / ml aprotinin. Insoluble pieces were removed by centrifugation (20,190 × g, 2 hours, 4 ° C.). All additional steps in the purification were carried out at 4 ° C. (results summarized in Table 3). The pH 10.2 extract was diluted to 1800 mL with 1 mM PMSF (to reduce Na 2 CO 3 to 0.075 M). Dilutions were then loaded onto a column of fresh Q-Sepharose (Pharmacia Biotech, Piscataway, NJ) equilibrated to 0.075 M Na 2 CO 3 , pH 10.2. After loading, the column was washed with 1) 0.075 M Na 2 CO 3 , pH 10.2 until the A 280 absorbance of the eluent reached the background; 2) wash with 10 mM NaHCO 3 , pH 8.5 until the pH of the melt is reduced to 8.5; 3) washed with 50 ml 10 mM NaHCO 3 , pH 8.5, 0.15 M NaCl; 4) washed with a 100 ml gradient of 0.15 M to 1.5 M NaCl in 10 mM NaHCO 3 , pH 8.5; 5) washed with 150 ml 10 mM NaHCO 3 , pH 8.5, 1.5 M NaCl; 6) washed with 10 mM NaHCO 3 , pH 8.5; 7) washed with 0.1 M Na 2 CO 3 , pH 11 until the pH of the precipitate rose to 11. Finally, uric acid enzyme was eluted with a 500 ml concentration gradient of NaCl 0 to 0.6 M in 0.1 M Na 2 CO 3 , pH 11. Activity eluted at the A 280 -absorption peak, which was collected separately (fraction A and fraction B, Table 3). The uric acidase in each fraction was then precipitated by slow addition of 1 M acetic acid to pH 7.1 and centrifugation (7,000 × g, 10 min). The resulting precipitate was dissolved in 50 mL of 1 M Na 2 CO 3 , pH 10.2 and stored at 4 ° C. Recombinant Pig-Bib Chimera (PBC) Uricase Purification IPTG-induced cells = 189.6 g FractionTotal protein mgUricase activity U / mlTotal Uricase Enzyme UnitSpecific activity U / mg pH 7 sonic debris + pH 7 washes 74.9pH 10.2 Extract471282.711,1702.4 Q-Sepharose fraction A fraction B82018.911.531.71,081 * 4,0801.92.3 pH 7.1 Precipitation and Redissolution Fraction A Fraction B598158635.075.51,7483,7733.02.4 Total yield2184 5,521 Uricase in fraction A precipitates naturally after eluting from the column. Therefore, the activity measured in the purification step is estimated to be lower than actual. Example 3 Small amount preparation and PEGylation of recombinant PBC urease This example shows that purified recombinant PBC urease can be used to prepare PEGhk urease. In this reaction, all urate dehydrogenase subunits are modified (lane 7 in FIG. 1), maintaining about 60% catalytic activity. A. Small Expression and Isolation of PBC Uricase (Table 4. FIG. 1) A 4 L culture of E. coli BL21 (DE3) pLysS transformed with pET3d-PBC cDNA was incubated in a rotary shaker (250 rpm) at 37 ° C. At the time point when OD 525 reached 0.7, cultures were induced with IPTG (0.4 mM, 6 hours). Cells were then collected and frozen at -20 ° C. Cells (15.3 g) were disrupted by freezing and thawing and extracted with 1 M Na 2 CO 3 , pH 10.2, 1 mM PMSF. After centrifugation (12,000 × g, 10 min, 4 ° C.), the supernatant (85 mL) was diluted 1:10 with water and loaded onto a Q-Sepharose column in a similar manner to Example 1. Uricase obtained from the above procedure was concentrated by pressure ultrafiltration using a PM30 membrane (Amicon, Beverly, MA). The concentrate was loaded onto a Sephacryl S-200 (Pharmacia Biotech, Piscataway, NJ) column (2.5 × 100 cm) equilibrated to 0.1 M Na 2 CO 3 , pH 10.2. Fractions showing uric acid enzyme activity were collected and concentrated by the pressure filtration method described above. B. PEGylation Separable Separcryl S-200 PBC Uricase (5 mg / mL, 2.9 μmol Enzyme; 84.1 μmol Lysine) in 0.1 M Na 2 CO 3 , pH 10.2 2 quantitatively 2 times (PEG mol: uric acid lysine) mol) with active PEG at 4 ° C. for 60 minutes. PEGylated urateases were either unreacted or separated from hydrolyzed PEG by flow diafiltration of the tangent. In the process, the reaction with 0.1 M Na 2 CO 3, pH 10.2, and then with a 1:10 dilution, 3.5 vol 0.1 M Na 2 CO 3, followed by pH 10.2 3.5 vol 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2 Diamond filtration was performed. The filter-sterilizing enzyme was stable for at least 1 month at 4 ° C. Purification and PEGylation of Recombinant Porcine-Bubi Chimera (PBC) Uricase A. Purification FractionTotal protein mgTotal Uricase Activity μmol / minSpecific activity μmol / min / mg% Of activity Crude Extract156510100.6100 Q-Sepharose35510513.0104 Sephacryl S-20021511705.5116 B. PEGylation S-200 Uricase1005465.5100 PEG-Uricase973363.562 1 shows the SDS-mercaptoethanol PAGE (12% gel) analysis of the fractions obtained during purification and PEGylation of recombinant pig-bibi chimera (PBC) uratease. Lane: 1 = MW marker; 2 = SDS extract of uninduced pET3d-PBC cDNA-transformed cells (E. coli BL21 (DE3) pLysS); 3 = SDS extract of IPTG-induced pET-PBC cDNA-transformed cells; 4 = crude extract (see Table 5); 5 = concentrated Q-Sepharose uricase extract; 6 = concentrated Sephacryl S-200 Uricase Extract; 7 = PEGylated Sephacryl S-200 Recombinant PBC Uricase. The results in Table 4 show that the purified PBC urate dehydrogenase can be modified while maintaining about 60% catalytic activity. In the PEGylation reaction, all urate dehydrogenase subunits were modified (FIG. 1, lane 7). Although the results are not shown, PEGylated enzymes exhibit similar kinematic properties to unmodified PBC uratease (K M 10-20 μM). Importantly, the modified enzyme is more soluble at physiological pH (> 5 mg / ml in PBS vs. <1 mg / ml) compared to the unmodified enzyme. In addition, PEGylated enzymes were lyophilized with minimal loss of activity and could be reconstituted in PBS, pH 7.2. In another experiment, we compared the activity of PEG-PBC urease and A. flavus urease clinical preparations. At borate buffer pH 8.6, the A. flavus enzyme showed 10-14 times higher V max and 2 times higher K M. However, at PBS pH 7.2, PEG-PBC and unmodified fungi enzymes showed a <2 fold difference in uricase activity. Example 4 Cyclic Lifespan of Unmodified and PEGylated PBC Uricase in Mice 2 shows the circulating lifespan of unmodified and PEGylated PBC urease. One group of unmodified (circular) or PEG-modified (square) recombinant PBC urease (prepared in Example 3) was intraperitoneally injected into the group of mice (3 per time point). At the indicated times, blood was obtained from three mice to measure serum urate enzyme activity. PEGylated uratease (described in Example 3) exhibited circulating half-life of about 48 hours, whereas unmodified enzyme showed <2 hours (FIG. 2). Example 5 Efficacy of PEGylated Uratease of the Present Invention Figure 3 shows the relationship between the uric acid enzyme activity of serum and uric acid concentration of serum and urine. In this experiment, 0.4 IU PEGylated recombinant PBC urease was injected twice at 0 and 72 hours in homozygous urease-deficient knockout mice (Wu et al. 1994). Uricase deficient knockout mice were used in this experiment, unlike normal mice with uricase enzymes, knockout mice have a high concentration of uric acid in the blood and body fluids, similar to humans, to excrete large amounts of uric acid by urine. Because. Such high concentrations of uric acid cause severe damage to the kidneys of mice, which are often life threatening (Wu et al. 1994). The experiment shown in FIG. 3 shows that the urinary oxidase activity in serum was increased when intraperitoneally administered the PEGylated preparation of recombinant PBC urease, which significantly increased serum and urinary uric acid concentrations in urease-deficient mice. Decreases. Example 6 Non-immunogenicity of the Construct-carrier Complex Homozygous urease-deficient mice were repeatedly injected with PEGylated recombinant PBC urease without inducing promotion of clearance corresponding to the absence of significant immunogenicity. The result was confirmed by ELISA. 4 shows the maintenance of the circulating amount of uricase activity (measured in serum) after repeated injections. PEGylated PBC uricase was intraperitoneally injected at 6-10 day intervals. Serum urate enzyme activity was determined 24 hours after injection. Example 7 Covalent Bond to Lysine Introduced by Mutation PEGylation of purified recombinant PBC uratease binds to novel lysine (residue 291). In this experiment, PBC uratease could be modified by PEGylation. Whether the peptide comprising the novel lysine (residue 291) has been PEGylated modified can be determined by methods known to those skilled in the art. Reference Abuchowski A, Kazo GM, Verhoest CR, Jr., van Es T, Kafkewitz D, Nucci ML, Viau AT et a (1984) Cancer therapy with modified enzymes. I. Antitumor properties of polyethylene glycol-asparaginase conjugates. Cancer Biochem Biophys 7: 175-186 Abuchowski A, McCoy JR, Palczuk NC, van Es T, Davis FF (1977a) Effect of attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J Biol Chem 252: 3582-3586 Abuchowski A, van Es T, Palczuk NC, Davis FF (1977b) Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem 252: 3578-3581 Ausubel FM (1998) Current Protocols in Molecular Biology John Wiley ＆ Sons Ahn KJ, KIM YS, Lee HC, Park K, Huh KB (1992) Cyclosporine-induced hyperuricemia after renal transplant: Clinical Characteristics and mechani. Transplantation Proceedings 24: 1391-1392 Arellano F, Sacristan JA (1993) Allopurinol hypersensitivity syndrome: A review. Ann Pharmacother 27: 337-343 Becker MA (1988) Clinical aspects of monosodium urate monohydrate crystal deposition disease (gout). Rheumatic Disease Clinics of North America 14: 377-394 Becker MA, Roessler BJ (1995) Hyperuricemia and gout. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabolic and Molecular Bases of Inherited Disease, 7 th ed. McGraw-Hill, New York, pp 1655-1677 Brogard JM, Coumaros D, Frankhauser J, Stahl A, Stahl J (1972) Enzymatic uricolysis: A study of the effect of a fungal urate-oxydase. Eur J Clin Biol Res 17: 890-895 Brogard JM, Stahl A, Stahl J (1978) Enzymatic uricolysis and its use in therapy. In: Kelley WN, Arnold WJ, Weiner Im (eds) Uric Acid, Springer-Verlag, New York, pp 515-524 Chaffee S, Mary A, Stiehm ER, Girault D, Fischer A, Hershfield MS (1992) IgG antibody response to polyethylene glycol-modified adenosine deaminase (PEG-ADA) in patients with adenosine deaminase deficiency. J Clin Invest 89: 1643-1651 Chen RHL, Abuchowski A, van Es T, Palczuk NC, Davis FF (1981) Properties of two urate oxidases modified by the covalent attachment of poly (ethylene glycol). Biochim Biophys Acta 660: 293-298 Chua CC, Greenberg ML, Viau AT, Nucci M, Brenckman WD, Jr., Hershfield MS (1988) Use of polyethylene glycol-modified uricase (PEG-uricase) to treat hyperuricemia in a patient with non-Hodgkin lymphoma. Ann Int Med 109: 114-117 Cohen LF, Balow JE, Magrth IT, Poplack DG, Ziegler JL (1980) Acute tumor lysis syndrome: A review of 37 patients with Burkitt's lymphoma. Am J Med 64: 468-491 Conley TG, Priest DG (1979) Purification of uricase from mammalian tissue. Preparative Biochemistry 9: 197-203 Davis FF, Kazo GM, Nucci ML, Abuchowski A (1991) Reduction of immunogenicity and extension of circulating life of peptides and proteins. In: Lee VHL (eds) Peptide and Protein Drug Delivery, Marcel Dekker, New York, pp831-864 Davis S, Abuchowski A, Park YK, Davis FF (1981a) Alteration of the circulating life and antigenic properties of bovine adenosine deaminase in mice by attachment of polyethylene glycol. Clin Exp Immunol 46: 649-652 Davis S, Park YK, Abuchowski A, Davis FF (1981b) Hypouricaemic effect of polyethylene glycol modified urate oxidase. Lancet 1: 281-283 Delaney V, Sumrani N, Daskalakis P, Hong JH, Sommer BG (1992) Hyperuricemia and gout in renal allograft recipients. Transplantation Proceedings 24: 1773-1774 Donadio D, Errera J, Navarro M, Izarn P (1981) Anaphylaxis-like manifestations after intravenous in jection of urate oxidase in an asthmatic child with acute leukemia (letter). Nouv Presse Med 10: 711-712 Erlich HA (1989) PCR Technology. Principles and applications for DNA amplification Stockton Press, New York Escudier B, Leclercq B, Tandonnet F, Nitenberg G (1984) Hyperuricemia resistant to urate oxidase. Efficacy of high doses (letter). Presse Med 13: 1340 Fam AG (1990) Strategies and controversies in the treatment of gout and hyperuricaemia. Balliere's Clinical Rheumatology 4: 177-192 George T, Mandell BF (1995) Gout in the transplant patient. J Clin Rheumatol 1: 328-334 Gold GL, Fritz BD (1957) Hyperuricemia associated wiht the treatment of leukemia Ann Int Med 47: 428-434 Greenberg ML, Hershfield MS (1989) A radiochemical-high-performance liquid chromatographic assay for urate oxidase in human plasma. Anal Biochem 176: 290-293 Harris JM, Zalipsky S (Ed.) (1997) Poly (ethylene glycol) Chemistry and Biological ApplicationsACS, Washington, DC Hershfield MS. (1997) Biochemistry and immunology of poly (ethylene glycol) -modified adenosine deaminase (PEG-ADA). In: Harris JM, Zalipsky S (eds) Poly (ethlene glycol) Chemistry and Biological Applications, ACS, Washington, DC, pp 145-154 Hershfield MS (1995) PEG-ADA replacement therapy for adenosine deaminase deficiency: An update after 8.5 years. Clin Immunol Innumopathol 76: S228-S232 Hershfield MS (1996) Gout and uric acid metabolism. In: Bennett JC, Plum F (eds) Cecil Textbook of Medicine, XX ed. WB Saunders, New York, pp 1508-1515 Hershfield MS, Buckley RH, Greenberg ML, Melton AL, Schiff R, Hatem C, Kurtzberg J et al (1987) Treatment of adenosine deaminase deficiency with polyethylene glycol-modified adenosine deaminase. N Engl J Med 316: 589-596 Hershfield MS, Chaffee S, Koro-Johnson L, Mary A, Smith AA, Short SA (1991) Use of site-directed mutagenesis to enhance the epitope shielding effect of covalent modification of proteins with polyethylene glycol. Proc Natl Acad Sci USA 88: 7185-7189 Hershfield MS, Mitchell BS (1995) Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabllic and Molecular Bases of Inherited Disease, 7 th ed. McGraw-Hill, New York, pp1725-1768 Hershfield MS, Seegmiller JE (1976) Gout and the regulation of purine biosynthesis. In: Quagliariello E (eds) Horizons in Biochemistry and Biophysics, Addison-Wesley, Reading, MA, pp 134-162 Jones DP, Stapleton FB, Kalwinsky D, McKay CP, Kellie SJ, Pui CH (1990) Renal dysfunction and hyperuricemia at presentation and relapse of acute lymphoblastic leukemia. Med Pediatr Oncol 18: 283-286 Kelley WN, Fox IH, Pallela TD (1989) Gout and related disorders of purine metabolism. In: Kelley WN, Harris ED, Ruddy S, Sledge CG (eds) Textbook of Rheumatology, 3rd ed. WB Saunders, Philadelphia, pp1395-1448 Kissel P, Lamarche M, Royer R (1968) Modification of uricaemia and the excretion of uric acid nitrogen by an enzyme of fungal origin. Nature 217: 72-74 Kissel P, Schmitt J, Streiff F, Makuary G, Schmidt C, Toussain P (1972) L'urate oxydase: son interet dans la prevention des hyperuricemies therapeutiques en hematologie. Ann Med Nancy 11: 519-535 Lee CC, Wu X, Gibbs RA, Cook RG, Muzny DM, Caskey CT (1988) Generation of cDNA directed by amino acid sequence: Cloning of urate oxidase. Science 239: 1288-1291 Legoux R, Delpech B, Dumont X, Guillemot JC, Ramond P, Shire D, Caput D et al (1992) Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase. J Biol Chem 267: 8565-8570 London M, Hudson PM (1957) Uricolytic activity of purified uricase in two human beings. Science 125: 937-938 Masera G, Jankovic M, Zurlo MG, Locasciulli A, Rossi MR, Uderzo C, Recchia M (1982) Urate-oxidase prophylaxis of uric acid-induced renal damage in childhood leukemia. J Pediatr 100: 152-155 Montagnac R, Schillinger F (1990) Anaphylctic complication tied to intravenous injection of urate oxidase. Nephrologie 11: 259 Mourad G, Cristol JP, Chong G, Andary M, Mion C (1984) Role of precipitating antiurate oxidase andtibodies in urate oxidase-resistant hyperuricemia (letter). Presse Med 13: 2585 Potaux L, Aparicio M, Maurel C, Ruedas ME, Martin-Dupont CL (1975) Uricolytic therapy. Value of urate oxidase in the treatment of hyperuricemias. Nouv Presse Med 4: 1109-1112 Priest DG, Pitts OM (1972) Reaction intermediate effects on the spectrophotometric uricase assay, Analytical Biochemistry 50: 195-205 Pui C-H, Relling MV, Lascombes F, Harrison PL, Struxiano A, Mondesir J-M, Riberio RC et al (1997) Urate oxidase in prevention and treatment of hyperuricemia associated with lymphoid malignancies. Leukemia 11: 1813-1816 Reddy PG, Nemali MR, Reddy MK, Reddy MN, Yuan PM, Yuen S, Laffler TG et al (1988) Isolation and sequence determination of a cDNA clone for rat peroxisomal urate oxidase: Liver-specific expression in the rat, Proc Natl Acad Sci USA 85: 9081-9085 Rosenthal AK, Ryan LM (1995) Treatment of refractory crystal-associated arthtitis. Rheum Dis Clin North Amer 21: 151-161 Roubenoff R (1990) Gout and hyperuricemia. Rheumatic Disease Clinics of North America 16: 539-550 Sadler JR, Miwa J, Maas P, Smith T (1974) growth of high density bacterial cultures; a simple device. Laboratory Practice 23: 632-643 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Pages Sandberg AA, Cartwright GB, Wintrobe MM (1956) Studies on leukemia. I. Uric acid excretion. Blood 11: 154-166 Savoca KV, Davis FF, Palczuk NC (1984) Induction of tolerance in mice by Uricase and monomethoxypolyethylene glycol-modified uricase. Int Arch Allergy Appl Immunol 75: 58-67 Sibony G, North ML, Bergerat JP, Lang JM, Oberling F (1984) Hyperuricemia resistant to urate ozidase. Role of anti-serum urate oxidase precipitating antibodies (letter). Presse Med 13: 443 Singer JZ, Wallace SL (1986) The allopurinol hypersensitivity syndrome. Unnecessary morbidity and mortality. Arthritis Rheum 29: 82-87 Tsuji J, Hirose K, Kasahara E, Naitoh M, Yamamoto I (1985) Studies on the antigenicity of the polyethylene glycol-modified uricase. Int J Immunopharmacol 7: 725-730 Venkataseshan VS, Feingold R, Dikman S, Churg J (1990) Acute hyperuricemic nephropathy and renal failure after transplantation. Nephron 56: 317-321 Veronese FM, Caliceti P, Schiavon O (1997) New synthetic polymers for enzyme and liposome modification. In: Harris JM, Zalipsky S (eds) Poly (ethylene glycol) Chemistry and Biological Applications, ACS, Washington, DC, pp 182-192 West C, Carpenter BJ, Hakala TR (1987) The incidence of gout in renal transplant recipients. Am J Kidney Dis 10: 369-371 Wu X, Lee CC, Muzny DM, Caskey CT (1989) Urate oxidase: Primary structure and evolutionary implications. Proc Natl Acad Sci USA 86: 9412-9416 Wu X, Muzny DM, Lee CC, Caskey CT (1992) Two independent mutational events in the loss of urate oxidase. J Mol Evol 34: 78-84 Wu X, Wakamiya M, Vaishnav S, Geske R, Montgomery CM, Jr., Jones P, Bradley A et al (1994) Hyperuricemia and urate nephropathy in urate oxidase-deficient mice. Proc Natl Acad Sci USA 91: 742-746 Ziitoun R, Dauchy F, Teillaud C, Barthelemy M, Bouchard P (1976) Le traitement des hyperuricemies en hematologie par l'urate-oxydase et l'allopurinol. Ann Med Interne 127: 479-482 The above documents are incorporated herein by reference. <110> DUKE UNIVERSITY <120> URATE OXIDASE <130> 1579-267 <150> US 60 / 095,489 <151> 1998-08-06 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 915 <212> DNA <213> Artificial Sequence <220> <223> PBC CHIMERA <220> <221> CDS <222> (1) .. (912) <223> Description of Artificial Sequence: PBC CHIMERA <400> 1 atg gct cat tac cgt aat gac tac aaa aag aat gat gag gta gag ttt 48 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 gtc cga act ggc tat ggg aag gat atg ata aaa gtt ctc cat att cag 96 Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 cga gat gga aaa tat cac agc att aaa gag gtg gca act tca gtg caa 144 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 ctg act ttg agc tcc aaa aaa gat tac ctg cat gga gac aat tca gat 192 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 gtc atc cct aca gac acc atc aag aac aca gtt aat gtc ctg gcg aag 240 Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 ttc aaa ggc atc aaa agc ata gaa act ttt gct gtg act atc tgt gag 288 Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 cat ttc ctt tct tcc ttc aag cat gtc atc aga gct caa gtc tat gtg 336 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 gaa gaa gtt cct tgg aag cgt ttt gaa aag aat gga gtt aag cat gtc 384 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 cat gca ttt att tat act cct act gga acg cac ttc tgt gag gtt gaa 432 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 cag ata agg aat gga cct cca gtc att cat tct gga atc aaa gac cta 480 Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 aaa gtc ttg aaa aca acc cag tct ggc ttt gaa gga ttc atc aag gac 528 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 cag ttc acc acc ctc cct gag gtg aag gac cgg tgc ttt gcc acc caa 576 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 gtg tac tgc aaa tgg cgc tac cac cag ggc aga gat gtg gac ttt gag 624 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 gcc acc tgg gac act gtt agg agc att gtc ctg cag aaa ttt gct ggg 672 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220 ccc tat gac aaa ggc gag tac tca ccc tct gtg cag aag acc ctc tat 720 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 gat atc cag gtg ctc tcc ctg agc cga gtt cct gag ata gaa gat atg 768 Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met 245 250 255 gaa atc agc ctg cca aac att cac tac ttc aat ata gac atg tcc aaa 816 Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys 260 265 270 atg ggt ctg atc aac aag gaa gag gtc ttg ctg cca tta gac aat cca 864 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 tat gga aaa att act ggt aca gtc aag agg aag ttg tct tca aga ctg 912 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 tga 915 <210> 2 <211> 304 <212> PRT <213> Artificial Sequence <400> 2 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 <210> 3 <211> 915 <212> DNA <213> Artificial Sequence <220> <223> PKS CHIMERA <220> <221> CDS <222> (1) .. (912) <223> Description of Artificial Sequence: PKS CHIMERA <400> 3 atg gct cat tac cgt aat gac tac aaa aag aat gat gag gta gag ttt 48 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 gtc cga act ggc tat ggg aag gat atg ata aaa gtt ctc cat att cag 96 Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 cga gat gga aaa tat cac agc att aaa gag gtg gca act tca gtg caa 144 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 ctg act ttg agc tcc aaa aaa gat tac ctg cat gga gac aat tca gat 192 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 gtc atc cct aca gac acc atc aag aac aca gtt aat gtc ctg gcg aag 240 Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 ttc aaa ggc atc aaa agc ata gaa act ttt gct gtg act atc tgt gag 288 Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 cat ttc ctt tct tcc ttc aag cat gtc atc aga gct caa gtc tat gtg 336 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 gaa gaa gtt cct tgg aag cgt ttt gaa aag aat gga gtt aag cat gtc 384 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 cat gca ttt att tat act cct act gga acg cac ttc tgt gag gtt gaa 432 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 cag ata agg aat gga cct cca gtc att cat tct gga atc aaa gac cta 480 Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 aaa gtc ttg aaa aca acc cag tct ggc ttt gaa gga ttc atc aag gac 528 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 cag ttc acc acc ctc cct gag gtg aag gac cgg tgc ttt gcc acc caa 576 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 gtg tac tgc aaa tgg cgc tac cac cag ggc aga gat gtg gac ttt gag 624 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 gcc acc tgg gac act gtt agg agc att gtc ctg cag aaa ttt gct ggg 672 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220 ccc tat gac aaa ggc gag tac tcg ccc tct gtc cag aag aca ctc tat 720 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 gac atc cag gtg ctc acc ctg ggc cag gtt cct gag ata gaa gat atg 768 Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met 245 250 255 gaa atc agc ctg cca aat att cac tac tta aac ata gac atg tcc aaa 816 Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys 260 265 270 atg gga ctg atc aac aag gaa gag gtc ttg cta cct tta gac aat cca 864 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 tat gga aaa att act ggt aca gtc aag agg aag ttg tct tca aga ctg 912 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 tga 915 <210> 4 <211> 304 <212> PRT <213> Artificial Sequence <400> 4 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 <210> 5 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: baboon D3H <400> 5 Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu 85 90 95 Tyr Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 <210> 6 <211> 304 <212> PRT <213> baboon <400> 6 Met Ala Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu 85 90 95 Tyr Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 <210> 7 <211> 304 <212> PRT <213> pig <400> 7 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Arg Ile Thr Gly Thr Val Lys Arg Lys Leu Thr Ser Arg Leu 290 295 300 <210> 8 <211> 298 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PBC amino truncated <400> 8 Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe Val Arg Thr Gly Tyr Gly 1 5 10 15 Lys Asp Met Ile Lys Val Leu His Ile Gln Arg Asp Gly Lys Tyr His 20 25 30 Ser Ile Lys Glu Val Ala Thr Ser Val Gln Leu Thr Leu Ser Ser Lys 35 40 45 Lys Asp Tyr Leu His Gly Asp Asn Ser Asp Val Ile Pro Thr Asp Thr 50 55 60 Ile Lys Asn Thr Val Asn Val Leu Ala Lys Phe Lys Gly Ile Lys Ser 65 70 75 80 Ile Glu Thr Phe Ala Val Thr Ile Cys Glu His Phe Leu Ser Ser Phe 85 90 95 Lys His Val Ile Arg Ala Gln Val Tyr Val Glu Glu Val Pro Trp Lys 100 105 110 Arg Phe Glu Lys Asn Gly Val Lys His Val His Ala Phe Ile Tyr Thr 115 120 125 Pro Thr Gly Thr His Phe Cys Glu Val Glu Gln Ile Arg Asn Gly Pro 130 135 140 Pro Val Ile His Ser Gly Ile Lys Asp Leu Lys Val Leu Lys Thr Thr 145 150 155 160 Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp Gln Phe Thr Thr Leu Pro 165 170 175 Glu Val Lys Asp Arg Cys Phe Ala Thr Gln Val Tyr Cys Lys Trp Arg 180 185 190 Tyr His Gln Gly Arg Asp Val Asp Phe Glu Ala Thr Trp Asp Thr Val 195 200 205 Arg Ser Ile Val Leu Gln Lys Phe Ala Gly Pro Tyr Asp Lys Gly Glu 210 215 220 Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr Asp Ile Gln Val Leu Ser 225 230 235 240 Leu Ser Arg Val Pro Glu Ile Glu Asp Met Glu Ile Ser Leu Pro Asn 245 250 255 Ile His Tyr Phe Asn Ile Asp Met Ser Lys Met Gly Leu Ile Asn Lys 260 265 270 Glu Glu Val Leu Leu Pro Leu Asp Asn Pro Tyr Gly Lys Ile Thr Gly 275 280 285 Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 <210> 9 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PBC carboxy truncated <400> 9 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser 290 295 300 <210> 10 <211> 298 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PKS carboxy truncated <400> 10 Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe Val Arg Thr Gly Tyr Gly 1 5 10 15 Lys Asp Met Ile Lys Val Leu His Ile Gln Arg Asp Gly Lys Tyr His 20 25 30 Ser Ile Lys Glu Val Ala Thr Ser Val Gln Leu Thr Leu Ser Ser Lys 35 40 45 Lys Asp Tyr Leu His Gly Asp Asn Ser Asp Val Ile Pro Thr Asp Thr 50 55 60 Ile Lys Asn Thr Val Asn Val Leu Ala Lys Phe Lys Gly Ile Lys Ser 65 70 75 80 Ile Glu Thr Phe Ala Val Thr Ile Cys Glu His Phe Leu Ser Ser Phe 85 90 95 Lys His Val Ile Arg Ala Gln Val Tyr Val Glu Glu Val Pro Trp Lys 100 105 110 Arg Phe Glu Lys Asn Gly Val Lys His Val His Ala Phe Ile Tyr Thr 115 120 125 Pro Thr Gly Thr His Phe Cys Glu Val Glu Gln Ile Arg Asn Gly Pro 130 135 140 Pro Val Ile His Ser Gly Ile Lys Asp Leu Lys Val Leu Lys Thr Thr 145 150 155 160 Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp Gln Phe Thr Thr Leu Pro 165 170 175 Glu Val Lys Asp Arg Cys Phe Ala Thr Gln Val Tyr Cys Lys Trp Arg 180 185 190 Tyr His Gln Gly Arg Asp Val Asp Phe Glu Ala Thr Trp Asp Thr Val 195 200 205 Arg Ser Ile Val Leu Gln Lys Phe Ala Gly Pro Tyr Asp Lys Gly Glu 210 215 220 Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr Asp Ile Gln Val Leu Thr 225 230 235 240 Leu Gly Gln Val Pro Glu Ile Glu Asp Met Glu Ile Ser Leu Pro Asn 245 250 255 Ile His Tyr Leu Asn Ile Asp Met Ser Lys Met Gly Leu Ile Asn Lys 260 265 270 Glu Glu Val Leu Leu Pro Leu Asp Asn Pro Tyr Gly Lys Ile Thr Gly 275 280 285 Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 <210> 11 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PKS carboxy truncated <400> 11 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser 290 295 300
权利要求:
Claims (17) [1" claim-type="Currently amended] A mammalian recombinant uricase protein that has been modified to insert one or more lysine residues. [2" claim-type="Currently amended] The recombinant uricase protein of claim 1, wherein the recombinant protein is a chimeric protein comprising amino acid sequences of two or more mammals. [3" claim-type="Currently amended] According to claim 2, wherein the recombinant uric acid enzyme chimeric protein comprises a 304 amino acid sequence, 225 N-terminal portion of the 304 amino acid sequence corresponds to the amino acid sequence 1-225 of pig uric acid enzyme of swine The remaining 79 amino acid sequences of the 304 amino acid sequences correspond to the amino acid sequences 226-304 of the ubiquinate urate lyase. [4" claim-type="Currently amended] The amino acid sequence of claim 2, wherein the recombinant uric acid chimeric protein comprises 304 amino acid sequences, and 288 N-terminal portions of the 304 amino acid sequences correspond to amino acid sequences 1-288 of swine urate enzyme. The remaining 16 amino acid sequences of the 304 amino acid sequences correspond to amino acid sequences 289-304 of the urinary lyase. [5" claim-type="Currently amended] A recombinant uricase protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. [6" claim-type="Currently amended] An isolated and purified nucleic acid molecule encoding the recombinant uric acid enzyme of claim 1. [7" claim-type="Currently amended] An isolated and purified nucleic acid molecule encoding the recombinant uric acid enzyme of claim 3. [8" claim-type="Currently amended] An isolated and purified nucleic acid molecule encoding the recombinant urease of claim 4. [9" claim-type="Currently amended] An isolated and purified nucleic acid molecule encoding the recombinant uric acid enzyme of claim 5. [10" claim-type="Currently amended] An isolated and purified nucleic acid molecule of claim 9 having the nucleotide sequence of SEQ ID NO: 1. [11" claim-type="Currently amended] An isolated and purified nucleic acid molecule of claim 9 having a nucleotide sequence of SEQ ID NO: 3. [12" claim-type="Currently amended] A vector comprising the nucleic acid molecule of claim 1. [13" claim-type="Currently amended] A vector comprising the nucleic acid molecule of claim 9. [14" claim-type="Currently amended] A plunger cell comprising the vector of claim 12. [15" claim-type="Currently amended] A plunger cell comprising the vector of claim 13. [16" claim-type="Currently amended] A method of increasing a PEG binding site that is useful and harmless to a urease protein, the method comprising the step of inserting at least one lysine residue into the urease protein. [17" claim-type="Currently amended] A method of increasing a PEG binding site that is useful and harmless to a urease protein, the method comprising the step of inserting at least one lysine residue into the urease protein instead of the arginine residue.
类似技术:
公开号 | 公开日 | 专利标题 US20190008978A1|2019-01-10|Conjugates of an il-2 moiety and a polymer AU2012362141B2|2017-09-21|PH20 polypeptide variants, formulations and uses thereof US20170035896A1|2017-02-09|Polymer factor ix moiety conjugates Asthana et al.2003|Microbial L-asparaginase: A potent antitumour enzyme Pasut et al.2008|Anti-cancer PEG-enzymes: 30 years old, but still a current approach US8273339B2|2012-09-25|Polymer-based compositions and conjugates of antimicrobial agents CA2162366C|2009-10-27|Conjugation-stabilized polypeptide compositions CN101511390B|2013-04-24|Method for the production of conjugates of insulin-like growth factor-i and poly| US7323167B2|2008-01-29|Method of treatment with modified arginine deiminase EP0675727B1|2002-04-10|Glycolipid enzyme-polymer conjugates KR20050105497A|2005-11-04|Polymer-factor ⅷ moiety conjugates CN102573917B|2015-06-17|Pegylated L-asparaginase TWI418787B|2013-12-11|Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof US5891704A|1999-04-06|Method to produce high levels of methioninase EP0668933B1|2002-10-02|Use of methioninase as an antitumor agent in anti-methionine chemotherapy JP3779728B2|2006-05-31|Production of tissue factor activity inhibitors CN102161984B|2016-06-01|The method of separating tetramer uric acid oxydiazing enzyme US8901277B2|2014-12-02|Interferon alpha mutant and its polyethylene glycol derivative US7413735B2|2008-08-19|Pharmaceutical composition comprising arginine deiminase for inhibiting angiogenesis US20140044672A1|2014-02-13|Conjugates of a gm-csf moiety and a polymer CA2659081C|2018-01-16|Crystallized oxalate decarboxylase and use thereof ES2296750T3|2008-05-01|Muted form of arginina deiminasa. US6913915B2|2005-07-05|PEG-modified uricase JP2020058366A|2020-04-16|Denatured lecithin-cholesterol acyltransferase enzyme CN101735991A|2010-06-16|Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates
同族专利:
公开号 | 公开日 PL207369B1|2010-12-31| KR100841634B1|2008-06-26| DK1100880T3|2011-01-24| ZA200100974B|2002-06-26| JP2010158244A|2010-07-22| CN101280293B|2013-09-11| WO2000008196A2|2000-02-17| EP1100880A4|2002-04-17| IL197339D0|2011-07-31| AU5336599A|2000-02-28| AT483797T|2010-10-15| EP1100880B1|2010-10-06| AU766421B2|2003-10-16| CN101280293A|2008-10-08| CA2337967A1|2000-02-17| HK1125402A1|2014-06-06| CN1322243A|2001-11-14| US7056713B1|2006-06-06| CZ2001466A3|2001-07-11| WO2000008196A9|2000-07-13| BR9913360A|2001-07-03| PL346222A1|2002-01-28| EP1100880A2|2001-05-23| JP5721954B2|2015-05-20| HK1037214A1|2011-05-06| EP2277998A1|2011-01-26| HU0103205A3|2006-01-30| HK1153508A1|2017-07-21| ES2352451T3|2011-02-18| NZ509633A|2003-04-29| BR9913360B1|2013-09-24| IL141221D0|2002-03-10| PT1100880E|2011-01-13| CA2337967C|2011-11-01| CY1111001T1|2015-06-11| JP2002524053A|2002-08-06| DE69942834D1|2010-11-18| HU229774B1|2014-07-28| IL197339A|2015-02-26| CZ304223B6|2014-01-15| IL141221A|2010-06-16| EP2277998B1|2016-04-20| WO2000008196A3|2000-03-30| JP2013215199A|2013-10-24| RU2290439C2|2006-12-27| HU0103205A2|2001-12-28|
引用文献:
公开号 | 申请日 | 公开日 | 申请人 | 专利标题
法律状态:
1998-08-06|Priority to US9548998P 1998-08-06|Priority to US60/095,489 1999-08-05|Application filed by 듀크 유니버시티 2001-06-25|Publication of KR20010053633A 2008-06-26|Application granted 2008-06-26|Publication of KR100841634B1
优先权:
[返回顶部]
申请号 | 申请日 | 专利标题 US9548998P| true| 1998-08-06|1998-08-06| US60/095,489|1998-08-06| 相关专利
Sulfonates, polymers, resist compositions and patterning process
Washing machine
Washing machine
Device for fixture finishing and tension adjusting of membrane
Structure for Equipping Band in a Plane Cathode Ray Tube
Process for preparation of 7 alpha-carboxyl 9, 11-epoxy steroids and intermediates useful therein an
国家/地区
|