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    • 专利摘要:
    The present invention provides anti-obesity proteins that control adipose tissue when administered to a patient. Thus, these drugs allow patients to overcome obesity disorders and live a normal life with reduced risk of type 2 diabetes, cardiovascular disease and cancer.
    公开号:KR19980064258A
    申请号:KR1019970069938
    申请日:1997-12-17
    公开日:1998-10-07
    发明作者:죤마이클 빌스；로날드유진 챈스；제임스아더 호프만；론리쥬니어 밀리칸
    申请人:피터지.스트링거；일라이릴리앤드캄파니；
    IPC主号:
    专利说明:

    Anti-obesity protein
    The present invention relates to the field of human medicine, in particular to the treatment of obesity and obesity related diseases. More specifically, the present invention relates to anti-obesity proteins that control adipose tissue when administered to a patient.
    Obesity, particularly upper body obesity, is a common and very serious public health problem in the United States and around the world. Recent statistics have shown that over 25% of the US population and over 27% of the Canadian population are overweight [Kutzmarsky, Al. Kuzmarski, R. J., Amer. J. of Clin. Nutr. 55: 495 S-502S (1992); Leader, rain. May, Reeder, B. A., Can. Med. Assoc. J., 146: 2009-2019 (1992). Upper body obesity is a very dangerous factor for people with type 2 diabetes, as well as a risk factor for cardiovascular disease and cancer. The medical cost of obesity has recently been estimated to be 150,000,000,000 worldwide. The problem is getting serious enough to be drafted by a medical association to fight the ever-increasing fat hyperplasia in American society.
    Most of these obesity-induced pathologies may be responsible for symptoms associated with dyslipidemia, hypertension and insulin resistance. Many studies have shown that reduced obesity by diet and exercise significantly lowers these risk factors. Unfortunately, treatment with diet and exercise is not successful with up to 95% failure rate. This failure may be due to the fact that symptoms are deeply associated with genetically inherited factors that contribute to increased appetite, higher calorie food preferences, decreased momentum and increased fat-forming metabolism. Those who inherit these genetic characteristics indicate that they are likely to gain weight despite their efforts to overcome the symptoms. Therefore, there is a need for a pharmacological agent that corrects these hyperlipidemia disorders and enables physicians to successfully treat obese patients despite genetic inheritance.
    Physiologists have thought for years that when a mammal eats too much, it sends an overweight fat signal to the brain that the body is overweight, and then causes the body to eat less and burn more energy sources. Hervey, G. R., Nature 22: 629-631 (1969). This feedback model is supported by side-by-side experiments involving purified hormones that control hyperlipidemia.
    ob / ob mice are obese and diabetic models known to carry autosomal recessive properties linked to six intrachromosomal mutations. Recently, Zhang, Y. and his colleagues published a site cloning of the rat gene associated with this symptom (Zhang, Y. et al., Nature 372: 425-432 (1994)). This report discloses the genetic coding of a 167 amino acid protein with a 21 amino acid signal peptide expressed only in adipose tissue. Subsequently, rat obesity genes were cloned and expressed [Murakami, T. et al., Biochem. Biophys. Res. Comm. 209: 944-952 (1995). Now we want to describe the protein encoded by the ob gene as an over-fat controlling hormone.
    The protein encoded by the ob gene is pharmacologically active, but the physical properties of the protein are undesirable. For example, human proteins are likely to precipitate and aggregate in pharmaceutical formulations or under physiological conditions. Protein preparations that contain precipitates or can precipitate proteins after infusion increase the risk of eliciting the patient's immune response and can cause irritation at the site of infusion. Therefore, it is necessary to develop pharmacological reagents that show pharmacological activity and provide improved physical and chemical stability.
    We have found that the aggregation observed in natural human proteins is to some extent attributable to hydrophobic interactions at the surface of the protein, specifically at residues 100 and 138. The inventors have also found that replacing these positions with charged amino acids greatly reduces the tendency of the ob protein to aggregate and provides significantly improved pharmacological reagents. Thus, the present invention provides a biologically active obesity protein. This reagent allows patients to overcome their obesity disorders and live a normal life with more normalized conditions from the risk of type 2 diabetes, cardiovascular disease and cancer.
    Summary of the Invention
    The present invention has one or more of substitution of Grp, Asp, His, Lys or Arg of Trp at position 100 or substitution of Glu, Asp, His, Lys or Arg of Trp at position 138, and a protein of SEQ ID NO: 1 Academically acceptable salts.


    In this sequence,
    Xaa at position 22 is Asn or Ser;
    Xaa at position 28 is Gln or absent;
    Xaa at position 72 is Asn, Gln, Glu, or Asp;
    Xaa at position 73 is Val or Met.
    The present invention also provides a method of treating obesity or obesity related symptoms comprising administering the protein of SEQ ID NO: 1 to a mammal in need thereof.
    The invention also provides a pharmaceutical formulation comprising the protein of SEQ ID NO: 1 and one or more pharmaceutically acceptable diluents, carriers or excipients.
    The present invention also provides a protein of SEQ ID NO: 1 further having a leader sequence linked to the N-terminus of the SEQ ID NO: 1 protein. Such proteins are useful for their anti-obesity activity and as intermediates in the preparation of SEQ ID NO: 1 protein.
    The invention also provides a DNA encoding a protein of SEQ ID NO: 1 having a SEQ ID NO: 1 protein and a leader sequence.
    Further embodiments of the invention
    (a) transforming the host cell with DNA encoding a protein of SEQ ID NO: 1 or a protein of SEQ ID NO: 1 having a leader sequence;
    (b) culturing the host cell under conditions capable of expressing the protein;
    (c) recover the expressed protein; Randomly
    (d) a method of producing a sequence 1 protein comprising enzymatically cleaving a leader sequence to produce the protein of sequence 1.
    The invention also provides a protein for use in the manufacture of a protein for use in treating obesity or obesity related symptoms and a medicament for treating obesity or obesity related symptoms.
    1 is a graph showing the effect of sequence 2 protein on food consumption of ob / ob mice.
    2 is a graph showing the effect of the sequence 2 protein solution on the body weight of male ob / ob mice.
    3 is a graph showing the effect of sequence 3 protein on food consumption of ob / ob mice.
    4 is a graph showing the effect of the sequence 3 protein solution on the body weight of male ob / ob mice.
    5 is a graph showing changes in aggregate size over time.
    6 is a graph showing changes in aggregate size over time.
    In the spirit of the present invention as disclosed and claimed herein, the following terms and abbreviations are defined as follows.
    Base pairs (bp) represent DNA or RNA. The abbreviations A, C, G and T, when present in DNA molecules, correspond to the 5'-monophosphate form of nucleotides, (deoxy) denine, (deoxycytidine, (deoxy) guanine and (deoxy) thymine), respectively. The abbreviations U, C, G and T respectively correspond to the 5'-monophosphate form uracil, cytidine, guanine and thymine of nucleosides, respectively, when occurring within an RNA molecule. T or a pair of C and G. In a DNA / RNA heteroduplex, base pairs can represent a pair of T or U and A or a pair of C and G.
    FMOC is an abbreviation for 9-fluorenylmethoxycarbonyl.
    Immunoreactive protein (s) is a term used to collectively describe antibodies, fragments of antibodies capable of binding antigens derived from parent antibody molecules, and single chain polypeptide binding molecules [Bud, E. (Bird, E. R.), PCT Application No. PCT / US87 / 02208, International Publication No. WO 88/01649 (published March 10, 1988)]
    Plasmids are extracellular replicative genetic elements.
    PAM is an abbreviation of 4-hydroxymethylphenylacetamidomethyl.
    PMSF is an abbreviation for phenylmethylsulfonyl fluoride.
    The reading frame is a nucleotide sequence that is translated into triplets by the translation device of tRNA, ribosomes and related factors, corresponding to a particular amino acid. Because each triplet is distinct and has the same length, the coding sequence must be three-fold. Base pair insertion or deletion (called frame transfer mutations) can result in two different proteins encoded by the same DNA segment. To prevent this, triplet codons corresponding to the desired polypeptide should be aligned in multiples of the start codon. That is, the correct read frame must persist.
    Recombinant DNA cloning vectors consist of DNA molecules to which one or more DNA segments can be added or added with all spontaneous replication functional groups, including plasmids and phages.
    Recombinant DNA expression vectors are recombinant DNA cloning vectors into which a promoter has been introduced.
    TFA is an abbreviation for trifluoroacetic acid.
    Transcription refers to the process of transferring information retained in the nucleotide sequence of a DNA to a complementary RNA sequence.
    Translation is the process of specifying and directing the synthesis of polypeptide chains using the information of messenger dreams.
    Tris is an abbreviation for tris (hydroxymethyl) aminomethane.
    Treating refers to managing and protecting a patient to eliminate a disease, symptom, or condition, and to prevent the onset of symptoms or complications, administering a compound of the present invention to alleviate the symptoms or complications, or It includes removing. Therefore, treating obesity includes inhibiting food intake, inhibiting weight gain, and inducing weight loss in patients in need of obesity treatment.
    A vector is a replicon used for transformation of a cell in genetic engineering with a polynucleotide sequence that, when incorporated with the appropriate control sequence, imparts specific properties on the host cell to be transformed. Plasmids, viruses, and bacterial phages are suitable vectors because they are replicons under normal conditions. Artificial vectors are constructed by cleaving and incorporating DNA molecules from other sources using restriction enzymes and ligase. Vectors include recombinant DNA cloning vectors and recombinant DNA expression vectors.
    X-gal is an abbreviation of 5-bromo-4-chloro-3-indolyl beta-D-galatoside.
    Amino acid abbreviations are 37 C.F.R. Approved by the United States Patent and Trademark Office as described in §1.822 (b) (2) (1993).
    As noted above, the present invention provides a protein of SEQ ID NO: 1 having one or more of substitution of Trp at position 100 with Glu, Asp, His, Lys or Arg or substitution of Trp at position 138 with Glu, Asp, His, Lys or Arg To provide.
    SEQ ID NO: 1
    In this sequence,
    Xaa at position 22 is Asn or Ser;
    Xaa at position 28 is Gln or absent;
    Xaa at position 72 is Asn, Gln, Glu, or Asp;
    Xaa at position 73 is Val or Met.
    Preferred proteins of the invention are those wherein Xaa at position 22 is Asn in position 1, Xaa at position 28 is Gln, Xaa at position 72 is Asn or Asp, and Xaa at position 73 is Val.
    Other preferred proteins are proteins wherein the Trp at position 100 is replaced with Glu or Asp, or the Trp at position 138 is replaced with Glu or Asp. Particularly preferred proteins are those wherein Xaa at position 72 in SEQ ID NO: 1 is Asp. Other preferred proteins are those wherein the Trp at position 100 is replaced by His, Lys or Arg. Another preferred protein is a protein in which the Trp at position 100 is substituted with Lys or Arg or the Trp at position 138 is substituted with Lys or Arg.
    Most preferred proteins of the invention are proteins of the following sequence.




    The present invention provides a biologically active protein that effectively treats obesity. Applicants have found that certain substitutions for hydrophobic residues on the protein surface confer improved properties. These residues could not be predicted from the primary sequence. Substituted proteins are pharmacologically active and have a reduced tendency to aggregate when compared to both the murine and human forms of the protein. The present invention makes it easier to formulate obesity proteins at high concentrations and increases pharmaceutical precision because they are compatible with commonly used preservatives.
    The claimed proteins are usually produced by recombinant technology. Techniques for obtaining mutations by substitution at predetermined sites of DNA with known sequences are well known, for example M13 primer mutations. Mutations that can be made in the DNA encoding the anti-obesity protein of the present invention should arrange the sequence outside the reading frame and preferably do not form complementary regions capable of producing secondary mRNA structures. DeBoer, H.A., European Patent Publication No. 75,444 A2, published March 3, 1983].
    Compounds of the invention can be prepared by well known chemical procedures such as recombinant DNA techniques or solution or solid phase peptide synthesis, or semisynthesis in solution starting with bound protein fragments via conventional solution methods.
    A. Solid Phase
    Synthesis of the claimed proteins can proceed by solid phase peptide synthesis or by recombinant methods. The principles of solid phase chemical synthesis of polypeptides are well known in the art and are described by Dugas, H. and Penny, C. Bioorganic Chemistry Springer-Verlag, New York, (1981). 54-59, such as general literature in the art. For example, peptides were synthesized by a PE-Applied Biosystems 433A Peptide Synthesizer (Perkin Elmer-Applied Biosystems Division, Foster City, Calif.) And Applied Biosystems. It can be synthesized by the solid phase method using. Boc amino acids and other reagents can be purchased from PE-Applied Biosystems and other chemical suppliers. Continuous Boc chemistry using a double bond protocol is applied to the starting p-methyl benzhydryl amine resin to produce the C-terminal carboxamide. For the production of C-terminal acids the corresponding PAM resin is used. Arginine, asparagine, glutamine, histidine and methionine are bound using preformed hydroxy benzotriazole esters. The following side chain protection can be used.
    Arg, chubby
    Asp, cyclohexyl or benzyl
    Cys, 4-methylbenzyl
    Glu, cyclohexyl
    His, benzyloxymethyl
    Lys, 2-benzyloxycarbonyl
    Met, sulfoxide
    Ser, benzyl
    Thr, Benzil
    Trp, Formil
    Tyr, 4-bromo carbobenzoxy
    Boc deprotection can be performed using trifluoroacetic acid (TFA) in methylene chloride. Removal of formyl from Trp is carried out by treatment of the peptidyl resin with 20% piperidine in dimethylformamide for 60 minutes at 4 ° C. Met (O) can be reduced by treatment with TFA / dimethylsulfide / conc. HCl (95/5/1) at 25 ° C. for 60 minutes. After this pretreatment, the peptide is further deprotected and contains a mixture of 10% m-cresol or m-cresol / 10% p-thiocresol or m-cresol / p-thiocresol / dimethyl sulfide Anhydrous hydrogen fluoride can be used to cut from the resin. Cleavage of the side chain protecting group (s) and cleaving the peptide from the resin are carried out at 0 ° C. or less, preferably at 30 ° C. for 30 minutes and then at 0 ° C. for 30 minutes. After removing HF, the peptide / resin is washed with ether. Peptides are extracted with glacial acetic acid and lyophilized. Purification was performed with reversed phase C18 chromatography in 0.1% TFA with increased gradient of acetonitrile, e.g. 2.2 cm x 25 cm Vydac column (The Separation Group, Hesperia, Calif.) The Separations Group, Inc.).
    Those skilled in the art will appreciate that solid phase synthesis can also be carried out using the FMOC method and the TFA / scavenger cleavage mixture.
    B. Recombinant Synthesis
    The claimed proteins can also be produced by recombinant methods. Recombinant methods are preferred when high yields are desired. Fundamental steps in recombinant production of proteins
    a) constructing synthetic or semisynthetic (or isolation from natural sources) DNA encoding the claimed protein,
    b) constructing the coding sequence alone or in an expression vector in a manner suitable for expression of the protein as a fusion protein,
    c) transforming the appropriate eukaryotic or prokaryotic host cell with an expression vector, and
    d) recovering and purifying the protein produced by recombination.
    a. Gene composition
    Synthetic genes that produce proteins by in vitro or in vivo transcription can be constructed by techniques well known in the art. Due to the degeneracy of the genetic code, those skilled in the art will understand that a DNA sequence encoding the claimed protein can be constructed in a significant but limited number. In a preferred implementation of the invention, the synthesis is carried out by recombinant DNA techniques.
    Methodologies of synthetic gene construction are well known in the art [Brown, E. Brown, E. L., Methods in Enzymology, Academic Press, New York, NY, 68: 109-151 (1979). DNA sequences corresponding to the synthesized claimed protein genes can be formed using conventional DNA-compositing devices such as Applied Biosystems Model 380A or 380B DNA synthesizers (Perkin Elmer, Applied Biosystems Division, Proster, Calif.) have.
    In some cases, it may be desirable to modify the coding sequence of the claimed protein to introduce a convenient protease sensitive cleavage site, such as a structural protein that facilitates controlled cleavage of the signal peptide from the signal peptide and the fusion protein construct.
    Genes encoding the claimed proteins can also be formed by using polymerase chain reaction (PCR). The template may be cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from human adipose tissue. Such methods are well known in the art. For example, Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).
    b. Direct expression or fusion protein
    The claimed protein can be prepared by direct expression or by cleavage as a fusion protein comprising the claimed protein followed by enzymatic or chemical cleavage. Various peptidases are known that cleave polypeptides at specific sites or digest amino or carboxy termini of peptide chains. In addition, certain chemicals (eg, cyanogen bromide) cleave the polypeptide at specific sites. Those skilled in the art will understand the modifications necessary for amino acid sequences (and synthetic or semisynthetic coding sequences when using recombinant means) to introduce site specific internal cleavage sites. See, eg, Carter, P., Protein Purification, Chapter 13: From Molecular Mechanisms to Large-Scale Processes, Ladisch, M., Eds. American Chemical Soc., Washington, D.C. (1990)].
    c. Vector composition
    Standard ligation techniques are used to construct suitable vectors with the desired encryption and control sequences. Isolated plasmids or DNA fragments are cleaved, adjusted and re-ligated to the desired form to form the required plasmid.
    In order to perform translation of the desired protein, synthetic DNA sequences which have been subjected to appropriate recombinant DNA expression vector excess by inserting appropriate restriction endonucleases are inserted. Synthetic coding sequences are designed to have restriction endonuclease cleavage sites at both ends of transcription that facilitate these expressions and amplification and isolation from expression plasmids and fusion to said plasmids. Isolated cDNA coding sequences can be readily modified by the use of synthetic linkers to facilitate incorporation of these sequences into the desired cloning vector by techniques well known in the art. The specific endonuclease used is searched by the restriction endonuclease cleavage pattern of the parental expression vector to be used. Restriction sites are chosen to properly orient the coding sequence with the control sequence to realize appropriate in-frame reading and expression of the claimed protein.
    Generally, plasmid vectors containing promoters and control sequences derived from species compatible with the host cell are used with the host cell. Vectors usually involve replication sites as well as marker sequences that can provide phenotypic selection in transformed cells. For example, this. Collie is typically this. Transformation is carried out using pBR322, a plasmid derived from Coli species (Bolivar, F.), Gene 2: 95-113 (1977). Plasmid pBR322 provides an easy means to identify transformed cells with genes for ampicillin and tetracycline resistance. The pBR322 plasmid or other microbial plasmid should also be modified to contain or contain promoters and other control elements commonly used in recombinant DNA techniques.
    The desired coding sequence is inserted into the expression vector in a suitable orientation to transcrib from a promoter and ribosomal binding site that can act in the host cell in which the protein is to be expressed. Examples of such expression vectors are Belagase, al., Issued April 19, 1994. Belagaje, R. M., is a plasmid described in US Pat. No. 5,304,473, which is incorporated herein by reference. The gene encoding A-C-B proinsulin described in US Pat. No. 5,304,473 can be removed from plasmid pRB182 using restriction enzymes NdeI and BamHI. Genes encoding proteins of the invention can be inserted into the plasmid backbone on the NdeI / BamHI restriction fragment cassette.
    d. Prokaryotic expression
    In general, prokaryotes are used for the cloning of DNA sequences in constructing vectors useful in the present invention. For example, Lee. Collie K12 strain 294 (ATCC wp 31446) is particularly useful. Other microbial strains that may be used include E. coli. Collie B and Lee. Collie X1776 (ATCC 31537). These examples are illustrative and not limiting.
    Prokaryotes are also used for expression. As well as the aforementioned strains. Intestinal bacteria and various Pseudomonas species can be used, such as Colli W3110 (prototrophic, ATCC 27325), Bacillus such as Bacillus subtilis and Salmonella typhimurium or Serratia marcescan. Promoters suitable for use with prokaryotic host include β-lactamase (vector pGX2907 [ATCC 39344] contains replicon and β-lactamase genes) and lactose promoter system [Chapter, A. Seed. Chang, A. C. Y. et al., Nature, 275: 617-624 (1978); And Godel, D. Goeddel, DV all, Nature 281: 544-548 (1979)], alkaline phosphatase, tryptophan (trp) promoter system (vector pATH1 [ATCC 37695]) expresses an open reading frame as a trpE fusion protein under the control of the trp promoter. Hybrid promoters such as tac promoter (isolated from plasmid pDR540 ATCC-37282). However, one of ordinary skill in the art can ligation to DNA encoding a protein using a linker or adapter to which bacterial promoters of other functions, generally known as nucleotide sequences, supply any necessary restriction sites.
    e. Eukaryotic Cell Expression
    Proteins can be produced recombinantly in a eukaryotic cell expression system. Preferred promoters for controlling transcription in mammalian host cells include a variety of sources, such as polyomas, Simian Virus 40 (SV40), adenoviruses, retroviruses, hepatitis-B viruses and most preferably cytomegaloviruses. From the genome of the same virus or from heterologous mammalian promoters such as the β-actin promoter. The early and late promoters of the SV40 virus are readily obtained as SV40 restriction fragments that also contain the SV40 viral base of replication (Fiers, W. et al., Nature, 273: 113-120 (1978)). The entire SV40 genome can be obtained from plasmid pBRSV, ATCC 45019. The first promoter of human cytomegalovirus can be obtained from plasmid pCMBβ (ATCC 77177). Of course, promoters from host cells or related species are also useful herein.
    The transcription of the DNA encoding the claimed protein by higher eukaryotic cells is increased by inserting a fortifying sequence into the vector. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp, that act on the promoter and increase its transcription. Enhancers encode the coding sequence itself [Osborn, T. Osborne, T. F., et al., Mol. Cell. Bio. 4: 1293-1305 (1984)] as well as within the Intron [Banergi, J., et al., Cell 33: 729-740 (1983)]. . Laimins, L. A., Proc. Nat'l Acad. Sci. (USA) 78: 464-468 (1981)] and 3 '(Lusky, M.), Mol. Cell. Bio. 3: 1108-1122 (1983) and are relatively orientation and position independent. Many enhancer sequences are currently known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, α-fectoprotein and insulin). However, one would usually use enhancers obtained from eukaryotic cell viruses. Examples include SV40 late enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers on the second half of origin of replication and adenovirus enhancers.
    Expression vectors used in eukaryotic host cells (nucleated cells from yeasts, fungi, insects, plants, animals, humans or other multicellular organisms) will also contain sequences necessary for the determination of transcription that can affect mRNA expression. . This region is transcribed into the polyadenylation segment in the untranslated portion of the mRNA encoding protein. The 3 'untranslated region also includes a transcription termination site.
    Expression vectors may contain a selection gene, also called a selectable marker. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (which can be derived from Bg1II / HindIII restriction fragments of DHFR, pJOD-10 [ATCC 68815]), thymidine kinase (simple herpes virus thymidine kinase) Is contained on the BamHI fragment of the neomycin (G418) restriction gene obtainable from the vP-5 clone (ATCC 2028) or the pNN414 yeast artificial chromosome vector (ATCC 37682). When such selectable markers are successfully delivered into mammalian host cells, the transfected mammalian host cells can survive if placed under selective pressure. There are two distinct categories of selective curing that are widely used. The first category is based on the metabolism of cells and uses mutant cell lines that cannot grow without supplemented medium. There are two examples. CHO DHFR - cells (ATCC CRL-9096) and murine LTK - cells [LM (TK-) ATCC CCL-2.3]. These cells cannot grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes essential for the intact nucleotide synthesis pathway, they cannot survive without missing nucleotides provided in supplemented medium. An alternative to supplementing the medium is to incorporate the complete DHFR or TK gene into cells lacking each gene to change the growth conditions. Individual cells not transformed with the DHFR or TK gene will not be able to survive in the medium not supplemented.
    The second category is the predominant selection representing the selection scheme used for any cell type. This scheme usually uses drugs to arrest the growth of host cells. Cells with new genes express protein delivery drug resistance and survive selection. Examples of such dominant selectivity include the drug neomycin [Southern P J., J. Molec. Appl. Genet. 1: 327-341 (1982)], mycophenolic acid [Muligan, R.. C. (Mulligan, RC), Science 209: 1422-1427 (1980)] or Hygromycin (Sugden, B.), Mol Cell Biol. 5: 410-413 (1985) The three examples given above use bacterial genes such as G418, neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin under eukaryotic cell control, respectively, that deliver resistance to the appropriate drug. do.
    Preferred vectors for eukaryotic cell expression are pRc / CMV. pRc / CMV is available from Invitrogen Corporation, San Diego, California. To ensure that the sequences in the plasmids are constructed correctly, the ligation mixture is used to determine the E. coli. Coli K12 strain DH5a (ATCC 31446) is transformed and successful transformants are appropriately selected by antibiotic resistance. Plasmids are prepared from the transformants and analyzed by restriction and / or sequenced by Messing, J. all methods (Nucleic Acids Res. 9: 309-321 (1981)).
    Host cells are incubated in conventional nutrient media modified to be suitable for inducing promoters upon transformation and for selecting transformants or for amplifying genes using the expression vectors of the invention. Culture conditions such as temperature, pH, etc., have been used prior to host cells selected for expression and will be apparent to those skilled in the art. Techniques for transforming cells with the vector are well known in the art and include maniatis, T. a. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), Current Protocols in Molecular Biology (1989), and supplements.
    Preferred host cells suitable for expressing vectors encoding the claimed proteins in higher eukaryotic cells include African green monkey kidney cell lines (COS-7, ATCC CRL-1651) transformed by SV40; Transform human early fetal kidney cell line 293 [Graham, F. Graham, FL, J. Gen Viro. 36: 59-72 (1977); Harrison, T., et al., Virology 77: 319-329 (1977); Graham, f. L. Eg, Virology 86: 10-21 (1978); Baby hamster kidney cells [BHK-21 (C-13), ATCC CCL-10; MacPherson, I., et al., Virologh 16: 147-151 (1962); Chinese hamster ovary cells [CHO-DHFR - (ATCC CRL -9096)]; Mouse Sertoli cells [TM4, ATCC CRL-1715; Matt, Jay. (Mather, JP), Biol. Reprod. 23: 243-252 (1980); African green monkey kidney cells (VERO 76, ATCC CRL-1587); Human cervical epithelial carcinoma cells (HeLa, ATCC CCL-2); Canine kidney cells (MECK, ATCC CCL-34); Murphyl rat liver cells (BRL 3A, ATCC CRL-1442); Human drainage chromosome lung cells (WI-38, ATCC CCL-75); Human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065); And murine breast cancer cells (MMT 060562, ATCC CCL51).
    f. Yeast expression
    In addition to prokaryotic mammalian host cells, eukaryotic microorganisms such as yeast may also be used as host cells. Many other strains are commonly available, but Saccharomyces cerbizier, a common baker's yeast, is the eukaryotic microorganism most commonly used to express heterologous proteins. In expression in Saccharomyces, for example, plasmid YRp7 is commonly used [ATCC-40053, Stinchcomb, D. Stinchcomb, D. T. et al., Nature 282: 39-43 (1979); Kingsman, a. Kingsman, A. J., et al., Gene 7: 141-152 (1979); Tschumper, G., Gene 10: 157-166 (1980). This plasmid already contains the trp gene, which provides a selection marker for mutant strains of yeast that cannot grow in tryptophan [eg, ATCC 44076 or PEP4-1; Jones, this. Jones, E. W., Genetics 85: 23-33 (1977).
    Facilitating sequences suitable for use in yeast hosts include the 3-phosphoglycerate kinase found in plasmid pAP12BD ATCC 53231 [Patel, A. (Patel, AC,), US Pat. No. 4,935,350, issued June 19, 1990] or other glycolytic enzymes such as enolase found in plasmid pAC1 (ATCC 39532), plasmid pHcGAPC1 (ATCC 57090, 57091) glyceraldehyde-3-phosphate dehydrogenase, Zimomonas mobilis [Ingram, L. (Ingram, LO), US Patent No. 5,000,000, issued March 19, 1991], hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-force Poglycerate mutases, pyruvate kinases, triocellular phosphate isomerase, phosphoglucose isomerase and glucokinase.
    Other yeast promoters, which are inducible promoters with the additional advantage of transcription controlled by growth conditions, include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradable enzymes involved in nitrogen metabolism, plasmid vector pCL28XhoLHBPV [ATCC 39475; ready. V. Found in Metallothionein, Glyceraldehyde 3-phosphate dehydrogenase and plasmid pRY121 (ATCC 37658) contained in Red, VB et al., US Pat. No. 4,840,896, issued June 20, 1989. Promoter region for enzymes participating in the use of maltose and galactose, such as the GAL1 promoter. Suitable vectors and promoters for use in yeast expression are those described by Buttzmann, Al. Hitzeman, R. A. is further described in European Patent Publication No. 73,657A1 (published March 9, 1983). It is advantageous to use a yeast enhancer with a yeast promoter, such as UAS Gal from Saccharomyces cerbizier found with the CYC1 promoter on plasmid YEpsec--hlIbeta (ATCC 67024).
    The following examples are described to further illustrate the preparation of the claimed proteins. The scope of the invention should not be construed as consisting solely of the following examples.
    Example 1
    The gene of SEQ ID NO: 13 is assembled from -220 base pair and -240 base pair segments derived from chemically synthesized oligonucleotides.
    A 220 base pair segment extends from the NdeI site to the XbaI site at position 220 in the coding region and assembles from seven overlapping oligonucleotides ranging in length from 34 to 83 bases. A 240 base pair segment extending from XbaI to the BamHI site is also assembled from seven overlapping oligonucleotides ranging from 57 to 92 bases in length.
    To assemble these fragments, each of the seven oligonucleotides is mixed in equimolar amounts, typically at a concentration of about 1 to 2 pmol / μl. All nucleotides, except for the 5'-terminal nucleotide of the segment, prior to assembly are phosphorylated in standard kinase buffer with T4 DNA kinase using conditions defined by the reagent supplier. The mixture is heated to 95 ° C. and slowly cooled to room temperature for 1 to 2 hours to moderately slow recover the oligonucleotide. Oligonucleotides are then ligated with each other with a suitable cloning vector such as pUC18 or pUC19 using T4 DNA ligase. Buffers and conditions are recommended by the enzyme supplier. Vectors for 220 base pair fragments are digested with NdeI and XbaI, while vectors for 240 base pair fragments are digested with XbaI and BamHI before use. Using a ligation mixture. Coli DH10B cells (commercially available from Life Technologies, a producer of GIBCO / BRL products in Grand Island, NY) and transformed cells contain 100 μg / ml Ampicillin, X-gal and IPTG One trypton-east plate (TY, Difco, Detroit, Michigan) was covered. Overnight colonies are grown in liquid TY medium with 100 μg / ml of ampicillin and used for plasmid isolation and DNA sequencing. Plasmids with the correct sequence are preserved for complete gene combination. This is done by gel purification of 220 base pair and 240 base pair fragments and ligation of these two fragments with an expression vector such as pRB182 digested with A-C-B proinsulin and digested with NdeI and BamHI prior to use.
    Alternatively, the plasmid pET30 (Novagen, Madison, WI) is digested with NdeI and BamHI and the desired DNA sequence encoding the protein of the invention is known in the art and by the procedures described herein. The source of DNA is a synthetic oligonucleotide assembled by the methods known in the art and described herein.
    Example 2
    Plasmid pOJ722 (NRRL, B21,654) containing a DNA sequence encoding the desired protein was prepared in the same manner as in Example 1. Plasmids are digested with pm1I and Bsu36I. The recognition sequences of these enzymes are in the coding region for the proteins at nucleotide positions 275 and 360, respectively. Cloning vectors do not contain these cognitive sequences. Thus, only two fragments were observed, corresponding to the vector fragment after restriction enzyme digestion with Pm1I and Bsu36 and corresponding to ˜85 base pair fragments liberated from the protein coding sequence. This sequence was replaced with any DNA sequence encoding the amino acid substitution of the present invention. These DNA sequences were chemically synthesized as two oligonucleotides having complementary ends compatible with the ends generated by digestion with complementary bases and Pm1 and Bsu36I. The chemically synthesized oligonucleotides were mixed in equimolar amounts (1-10 pmol / μl), heated to 95 ° C. and slowly lowered to 20-25 ° C. temperature to restore slowly. Slow cooled oligonucleotides were used for standard ligation reactions. Ligation products. Transformed with Coli DH10B cells (GIBCO BRL) and transformed cells were grown in liquid TY medium with 10 μg / ml of tetracycline (Sigma, St. Louis, MO, and subjected to plasmid isolation and DNA sequencing Preserve the plasmid with the correct sequence.
    Example 3
    Expression plasmid pHS787 (protein of SEQ ID NO: 6)
    The pHS692 vector prepared in the same manner as in Example 1 was digested with Pm1I (New England Biolab, Beverly, Mass., 20 units) in New England Biolab buffer 1 at 373429 for 2 hours. Digestion was initiated by adjusting to England Biolab bugger 3 and adding 20 units of BstXI (New England Biolab), which proceeded for 2 hours at 55 ° C. The digest was alkaline phosphatase at 37 ° C. for 30 minutes (Bearinger, Indianapolis, Indiana). Treatment with 20 units of Boehringer Mannheim The resulting digest was purified on 1% agarose gel and 4170 bp fragments were isolated using the freeze compression method.
    The oligonucleotides 13824 and 13825 of SEQ ID NOs: 14 and 15 were subjected to 1 × kinase buffer, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 0.5 mM ATP, 1 mM DTT and 5 unit T4 polynucleotides at 37 ° C. for 30 minutes. Kinase in the presence of kinase (GIBCO BRL).
    5'-TGA GGC TTC CAG GAC TCC CCC CAG ACT GTC CAA TGT CTC CAG GCC ACT GGC GTC TGG CAA GTG GCA ACT TTT AGA GAA GGC CAG CAC-3 '
    5'-GTG CTG GCC TTC TCT AAA AGT TGC CAC TTG CCA GAC GCC AGT GGC CTG GAG ACA TTG GAC AGT CTG GGG GGA GTC CTG GAA GCC-3 '
    PmI / BstXI ligation pHS692 vector and 13824 and 13825 linkers were ligated overnight at 16 ° C. in the presence of 1 × Kinase buffer, 0.5 mM ATP and 1 unit T4 DNA ligase (GIBCO BRL). Ligation products. Colonies that were transformed with Coli BL21 (DE3) (NOVAGEN) and grown on TY plates supplemented with 10 μg / ml tetracycline were analyzed. Plasmid DNA was isolated and DNA sequence confirmed on a PE-Applied Biosystems 370 DNA sequencer. Plasmids containing the expected ˜400 bp NdeI to BamHI fragments were stored and named pHS787.
    Example 4
    Expression plasmid (protein of SEQ ID NO: 3)
    Plasmid pOJ722 was digested with PmII and Bsu36I. A synthetic DNA fragment of 5'-SEQ ID NO. 16, slow cooled to 5'-SEQ ID NO. 17, was inserted between the PmI and Bsu36I sites. After ligation, transformation and isolation, the sequences of the synthetic fragments were confirmed by DNA sequencing.
    Techniques for transforming cells with the aforementioned vectors are well known in the art. Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1988); Id., Current Protocols in Molecular Biology (1989) and additional evidence. E. used in the preferred embodiment of the invention exemplified herein. Techniques involved in the transformation of coli cells are well known in the art. Transformed E. Fine conditions for culturing E. coli cells are used. It depends on the nature of the collie host cell line and the expression or cloning vector. For example, a vector incorporating a heat-induced promoter-operator region, such as the c1857 heat-induced lambda-phage promoter-operator region, should convert the temperature from about 30 ° C. to about 40 ° C. in culture conditions to induce protein synthesis.
    In a preferred embodiment of the invention, Coli K12 RV308 cells are used as host cells but many other cell lines, such as E. coli. Coli K12 L210, L687, L693, L507, L640, L641, L695, L814 (E. coli B) are valid and not limited thereto. The transformed host cell is then plated on a suitable medium under selective pressure of antibiotics corresponding to the resistance gene present on the expression plasmid. The culture is then incubated at the appropriate time and temperature for the host cell line used.
    Proteins expressed in a high level bacterial expression system are agglomerated into granules or inclusions that characteristically contain high yields of overexpressed proteins [Kruger, J. Kreuger, J. K., Protein Folding, Gierasch, L. M. and King, J., des., American Association for the Advancement of Science Publication No. 89-18S, Washington, D. C., 136-142 (1990)]. Such protein aggregates must be dissolved and purified and isolated to the desired protein product [Kruger, J. K. All of these documents]. Various techniques are used to dissolve proteins using strong denaturing solutions such as guanidinium-HCl and / or weak denaturing solutions such as urea. The denaturing reagent in solution is progressively removed (often by dialisis) so that the denatured protein has its original structure. Specific conditions for denaturation and folding are determined by the particular protein expression system and / or protein in question.
    The protein of the present invention is preferably expressed in the leader sequence. Those skilled in the art will recognize that a variety of leader sequences may be used, but the leader sequence may be converted to Met-R 1 − (wherein the expressed protein may be readily converted to the protein claimed by cathepsin C or other suitable aminopeptidase). R 1 is all amino acids except Pro or absent). It is preferred that R is Arg, Ap or Tyr, and most preferably the protein is expressed in the Met-Arg leader sequence. It should be noted that the leader sequence does not significantly affect the stability or activity of the protein. However, the leader sequence is preferably cleaved from the protein. Thus, proteins having the sequence of Met-R 1 -SEQ ID NO 1 are useful as biological reagents and preferred as intermediates.
    Example 5
    The protein of SEQ ID NO: 3 having a Met-Arg leader sequence was obtained from E. coli. It was expressed in Collie and isolated and folded by the same technique as the previous example. The pH of the protein solution was lowered to 2.8. The Met-Arg leader sequence was cleaved by adding 6-10 milliunits of dDAP per mg protein (dDAP is a mucus fungus, Dictostere, described in US Pat. No. 5,565,330 to Atkinson, PR). Abbreviation of dipeptidylaminopeptidase isolated from Dicteostelium descoidium). The conversion reaction proceeds at room temperature for 2 to 8 hours. The progress of the reaction was determined by high performance reverse phase chromatography. The reaction was terminated by adjusting to pH 8 with NaOH. The Met-Arg protein was further purified by cation exchange chromatography in the presence of 7-8 M urea and size block chromatography in PBS. After final purification of the protein by size blocking chromatography, the protein was concentrated to 3 to 3.5 mg / ml in PBS.
    Purification of the claimed proteins is carried out by techniques known in the art and includes reverse phase chromatography, affinity chromatography, ion exchange and size blocking chromatography.
    The claimed protein contains two cysteine residues. Thus, disulfide bonds can be formed to stabilize the protein. The present invention encompasses Cys at position 96 with Cys at position 146 and the protein of SEQ ID NO: 1. Thus, disulfide bonds can be formed to stabilize the protein. The present invention includes proteins in which Cys at position 96 is crosslinked with 146 Cys, as well as proteins that do not have such disulfide bonds. Cys at 96 is preferably disulfide bonded to Cys at 146. In addition, the proteins of the present invention may exist in dimers, trimers, tetramers and other multimers, especially when formulated. These multimers are included within the scope of the present invention.
    The present invention provides a method of treating obesity. The method comprises administering to the organism an effective amount of an anti-obesity protein in an amount of about 1 to 100 μg / kg. Preferred amounts of use are about 10 to 100 μg / kg of active compound. The typical daily dosage for adults is about 0.5 to 100 mg.
    In the practice of this method, the compound of SEQ ID NO: 1 may be administered in a single dose or in multiple doses per day. Treatment regimens may require administration over an extended period of time. The quantity per dose administered or the total amount administered will be determined by the practitioner and will depend on the nature and the degree of the disease, the age and general health of the patient and the patient's resistance to the compound.
    The invention also provides pharmaceutical preparations comprising the compounds of the invention. Preferably, the pharmaceutically acceptable salt form of the protein can be formulated for parenteral administration for the treatment or prevention of obesity. For example, the compound of Fever 1 may be mixed with conventional carriers and excipients. The composition comprising the claimed protein contains about 0.1 to 95% by weight of the active protein, preferably in the form of a solution and more generally 10 to 30%. In addition, the proteins of the invention can be administered alone or in combination with other anti-obesity reagents or reagents useful for treating diabetes. For intravenous preparations, sterile preparations, preferably hydrochloride, are in the form of suitable solution salts of the SEQ ID NO: 1 protein, for example pharmaceutical diluents such as pyrogen-free water (distilled water), physiological saline or 5% glucose solution. It can be dissolved and administered within. Suitable soluble forms of the compounds can be prepared and administered as suspensions in aqueous or pharmaceutically acceptable oily substrates such as long chain fatty acids such as ethyl oleate. It is preferred to add alkylparabens, in particular pharmaceutically acceptable preservatives such as methylparaben, ethylparaben, propylparaben or butylparaben, or chlorobutanol to the formulation for use in multiple administrations. It is noted that the claimed proteins are also stable in the presence of phenolic preservatives such as m-cresol or phenol. The stabilization of the protein in the presence of phenolic preservatives provides for ischemia in pharmaceutical delivery, including improved preservative efficiency. The formulation is preferably prepared without salt to minimize the ionic strength of the formulation.
    Biological test
    Pathologic experiments suggest that proteins are released by peripheral fatty tissue and that these proteins can control weight gain in normal mice as well as in obese mice [Colman, D. Coleman, D. L., Diabetologia 14: 141-148 (1978). Therefore, the most closely related biological test involves injecting test water into several routes of administration, for example, intravenous (iv), subcutaneous (sc), intraperitoneal (ip) or small pumps or cannula Consumption, weight gain, plasma chemistry or hormones (glucose, insulin, ACTH, corticosterone, GH, T4) are monitored for several hours.
    Suitable test animals include normal mice (ICR, etc.) and obese mice (ob / ob, Avy / a, KK-Ay, chubby mice or fat mice). Ob / ob rat models of obesity and diabetes are generally recognized as having obesity symptoms in the art. A control of the nonspecific effect on this infusion is the active reagent itself in animals (db / db mice, fa / fa or cp / cp mice) that are believed to lack active reagents or receptors of similar compositions in the same animal monitoring the same variables. This is done with or without a vehicle. Proteins that are active in these models will show similar activity in other animals, especially humans.
    If food intake and liposynthesis are controlled, the target tissue is expected to be in the hypothalamus, so similar models inject the test directly into the brain, for example through the lateral or third ventricle (icv) or Or directly into certain hypothalamic nuclei such as arches, cerebral arch nuclei or atrioventricular nuclei. Such variables can be measured or the release of neurotransmitters known to control intake or metabolism can be monitored (eg NPY, galanine, norepinephrine, dopamine, β-endorphin release).
    Representative proteins described in Examples 6 and 7 are prepared according to the disclosure and examples provided herein. Met-Arg- indicates that the protein was prepared and tested by attaching a Met-Arg leader sequence. The amino acid sequence of the protein of the example was confirmed by mass spectrum and / or amino acid analysis. Proteins were folded with Cys residues that were crosslinked by disulfide bonds when tested. The ability of the protein of the invention to treat obesity in ob / ob mice is shown in Table 1-3. Similar studies can be performed ex vivo using hypothalamic tissue isolated within a ventricular fusion or tissue bath system. In this case, the release or electrophysical change in neurotransmitter is monitored.
    Example 6
    Test of Ob / ob Male Rats with Ob Proteins Having SEQ ID NOs: 2, 3, and 6
    Male ob / ob rats (Harlan, Ltd., Blackton, England) were bred in groups of 5 each and provided with Purina 5008 food and water from time to time. Mice were kept in a backlight system (lighted off at 9:00 am and lit at 9:00 pm). The rat model was weighed at 8:30 am daily. Their food and water consumption was measured at the same time. As shown below, treatment was weighed just before the light went out. Mice were treated once for 4 days.
    groupTreatment (n = 5) OnePBS 0.2 ml / day, s.c. 2Protein 30 μg / 0.2 mL / day, s.c. 3Protein 300 μg / 0.2 mL / day, s.c.
    The effect of treatment on food consumption and cumulative weight change is shown in Tables 1-3 for representative proteins of the invention.
    Effect of Sequence 2 Protein on Food Consumption and Cumulative Weight Changes in ob / ob RatsFood consumption (g / day)Weight change (g) Work1 group2 group3 group1 group2 group3 group One4.44.12.4+0.5-0.6-0.8 25.34.33.0+0.4-0.2-1.3 34.84.22.4+0.6-0.2-2.3 45.13.81.5+0.9-0.6-3.5
    Effect of Sequence 3 Protein on Food Consumption and Cumulative Weight Changes in ob / ob RatsFood consumption (g / day)Weight change (g) Work1 group2 group3 group1 group2 group3 group One5.64.94.0+0.3-0.2-0.5 26.14.03.1+0.5-0.3-1.0 35.93.52.3+0.6-0.5-2.0
    Effect of Sequence 6 on Food Consumption and Cumulative Weight Changes in ob / ob RatsFood consumption (g / day)Weight change (g) Work1 group2 group3 group1 group2 group3 group One6.24.64+0.4-0.1-0.7 26.43.93.5+0.4-0.3-0.9 35.53.42.1+0.5-0.6-1.7 45.52.81.6+0.4-1.3-2.5 Group 1 = control group (PBS); Group 2 = 30 μg / day of protein; Group 3 = 100 μg / day protein
    Example 7
    Aggregate Analysis by Dynamic Light Scattering (DLS)
    The physical properties of the compounds of the present invention are demonstrated as follows. Based on the effective materials, solutions analyzed by DLS were prepared in two ways. The material was supplied at approximately 1.5 mg / ml to the solution obtained from the final size block chromatography purification step in PBS of PBS and concentrated to 3-5 mg / ml by diafiltering or dialisising with water.
    In the first method, approximately 1.5 mg / ml protein solution was seeded to greater than 3-5 mg / ml in a small volume stirred cell (10 ml) using Amicon YM10, 25 mm membrane. This was done in cold room conditions (about 5 ° C.). Protein concentration was determined by UV absorption and the solution was diluted to 3.0 mg / ml using PBS (10-fold water dilution of 10 × PBS (Ca / Mg, no GIBCO BRL)).
    The second alternative method used for lyophilization is easy to implement and consists in dialising the protein solution against water using 3 to 5 exchanges of water at about 4 ° C. Representative dialyzes membranes are Spectra / Pro-7 dialyzes membranes, 2000 molecular weight cleavage membranes (Spectrum Medical Industries, Los Angeles, Calif.). The material was concentrated to 3-4 mg / ml as above and typically the 2 mg yield was lyophilized in a 5 ml glass bottle. For DLS analysis, this plug was reconstituted with water to greater than 3.3 mg / ml or greater than 5 mg / ml. Combined several glass bottles. Protein concentration was determined by UV at the maximum peak (usually 280 nm). The protein concentrate was diluted to about 3-5 mg / ml with a combination of water and 10 × PBS (Ca / Mg, no GIBCO BRL) to give the final 1 × PBS concentrate.
    A 3.0 mg / ml or 5 mg / ml protein solution in 1 × PBS was adjusted to 7.4 with HCl / NaOH and 0.1 μm Anotop ™ -10 filter (Watman International, Maidstone, England). Ltd.) The average cumulative particle size was passed through a Brookhaven BI900 DLS device (Brookhaven Instruments, Holtsville, NY) every 15 minutes with a period of 30 seconds at a 90 ° angle. Brookhaven Instruments) 488-nm filter with 400 μm pinholes was used Viscosity 0.6915 centipoise (cP) and refractive index 1.333 were used at 37 ° C. incubation temperature. It was calculated by the cumulative method using a baseline.
    Table 4 shows the measurement of the time it takes for the various anti-obesity protein compounds to reach an average light weight particle size of 50 nm in PBS solution at pH 7.4 and 37 ° C. The average light weight particle size was determined from cumulative analysis of the bimodal distribution consisting of monomers and high town populations. The time required for the average aggregate size to reach 50 nm was estimated by creating a coordinate of the size as a function of time. Proteins were folded with Cys residues crosslinked by disulfide bonds at the time of testing. For reference, the human Ob protein and the protein of SEQ ID NO: 18 are shown. The abbreviation u.d. indicates indeterminate and n.d. indicates indeterminate.
    Protein sequence 18 is as follows.
    Assessment of the time required for various anti-obesity proteins to reach an average light weight particle size of 50 nm in PBS solution at pH 7.4 and 37 ° C.Time to reach 50 nmdp (min) protein3 mg / ml5 mg / ml Man ob protein2u.d. SEQ ID NO: 2604n.d. SEQ ID NO: 357244 SEQ ID NO: 61356527 SEQ ID NO: 18124n.d.
    Compounds are active in one or more of these biological assays and are anti-obesity reagents. Thus, they are useful for treating obesity and diseases associated with obesity. However, proteins are not only useful as therapeutic reagents; Those skilled in the art will appreciate that they are useful in the production of diagnostic antibiotics and also as food additives in animals as proteins. Mammal weight control is required to improve weight extroversion in cosmetic applications.
    The principles, preferred embodiments and working modes of the invention have been described above. However, the invention to be protected herein should not be construed as limited to the specific forms disclosed, as such specific forms are to be considered as illustrative and not restrictive. Those skilled in the art can make modifications and variations without departing from the spirit of the invention.
    Administration of an anti-obesity protein having a sequence according to the invention can reduce food intake and inhibit weight gain.
    权利要求:
    Claims (6)
    [1" claim-type="Currently amended] At least one of Trp at position 100 with Glu, Asp, His, Lys, or Arg, or at position 138 with Trp at Glu, Asp, His, Lys, or Arg, optionally with the formula Met-R 1 (where R 1 is absent or has a leader sequence of any amino acid except Pro) or a pharmaceutically acceptable salt thereof.
    SEQ ID NO: 1


    In this sequence,
    Xaa at position 22 is Asn or Ser;
    Xaa at position 28 is Gln or absent;
    Xaa at position 72 is Asn, Gln, Glu, or Asp;
    Xaa at position 73 is Val or Met.
    [2" claim-type="Currently amended] The protein of claim 1, or a pharmaceutically acceptable salt thereof, having a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
    [3" claim-type="Currently amended] A protein or a pharmaceutically acceptable salt thereof comprising the protein claimed in claim 1 or 2 and a leader sequence linked to the N-terminus of the protein.
    [4" claim-type="Currently amended] A pharmaceutical formulation comprising the protein claimed in any one of claims 1, 2 or 3 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable diluents, carriers or excipients.
    [5" claim-type="Currently amended] A DNA polynucleotide compound comprising DNA encoding a protein as claimed in claim 1.
    [6" claim-type="Currently amended] (a) transforming the host cell with DNA encoding the protein;
    (b) culturing the transformed host cell to express the protein;
    (c) enzymatically cleaving the leader sequence of the optionally expressed protein to produce the protein as claimed in claim 1 or 2;
    (d) A method for producing a protein as claimed in any one of claims 1, 2 or 3, comprising recovering the protein.
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    同族专利:
    公开号 | 公开日
    AU4843297A|1998-06-25|
    TR199701666A3|1999-10-21|
    AU5384698A|1998-07-17|
    HU9702500A3|1999-07-28|
    CO4700343A1|1998-12-29|
    SV1997000107A|1999-01-18|
    NO975938D0|1997-12-17|
    IL122569D0|1998-06-15|
    SG65040A1|1999-05-25|
    WO1998028335A1|1998-07-02|
    CA2217698A1|1998-06-20|
    TR199701666A2|1999-10-21|
    PE47599A1|1999-05-14|
    CZ407097A3|1998-07-15|
    HU9702500A2|1998-07-28|
    EP0849276A1|1998-06-24|
    AR013886A1|2001-01-31|
    BR9706358A|1999-05-04|
    NZ329413A|1999-10-28|
    ZA9710877B|1999-06-03|
    JPH10179158A|1998-07-07|
    ID19257A|1998-06-28|
    YU48997A|1999-09-27|
    NO975938L|1998-06-22|
    CN1194986A|1998-10-07|
    HU9702500D0|1998-03-02|
    PL323956A1|1998-06-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1996-12-20|Priority to US3356196P
    1996-12-20|Priority to US60/033,561
    1997-12-17|Application filed by 피터지.스트링거, 일라이릴리앤드캄파니
    1998-10-07|Publication of KR19980064258A
    优先权:
    申请号 | 申请日 | 专利标题
    US3356196P| true| 1996-12-20|1996-12-20|
    US60/033,561|1996-12-20|
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