Claims for Patent: 6,037,145

Summary for Patent: 6,037,145

Title:	Process for production of protein
Abstract:	A process for the production of a desired polypeptide comprising the steps of: (1) transforming host cells with an expression vector comprising a gene coding for a fusion protein comprising a desired polypeptide and a protective polypeptide; (2) culturing the transformed host cells so as to express said gene to produce a fusion protein; and (3) excising the desired polypeptide from the fusion protein with a protease intrinsic to the host cells. According to the present invention, a large amount of a desired polypeptide can be produced at a low cost. Especially according to the present invention, a large amount of S. aureus V8 protease can be efficiently produced at low cost using a safe host such as E. coli according to gene recombination procedures.
Inventor(s):	Yabuta; Masayuki (Tatebayashi, JP), Ohsuye; Kazuhiro (Ohta, JP)
Assignee:	Suntory Limited (Osaka, JP)
Application Number:	08/523,373
Patent Claims:	1. A process for production of a desired polypeptide comprising the steps of: (1) transforming host cells with an expression vector comprising a gene coding for a fusion protein comprising a desired polypeptide and a protective polypeptide; (2) culturing the transformed host cells so as to express said gene to produce an insoluble fusion protein, which forms inclusion bodies, wherein said inclusion bodies further comprise a protease intrinsic to said host cells; and (3) excising the desired polypeptide from the fusion protein with said protease. 2. A process according to claim 1, wherein the fusion protein is represented by the formula (1) A-L-B, or (2) A-L-B-L-C, wherein A and C are protective polypeptides, B is a desired polypeptide and L is a linker peptide containing a substrate site specifically recognized by a protease intrinsic to the host cells, and the fusion protein is cleaved in the linker peptide L region so as to obtain the desired polypeptide B. 3. A process according to claim 2, wherein the desired polypeptide is a physiologically active polypeptide. 4. A process according to claim 3 wherein the physiologically active polypeptide is selected from the group consisting of motilin, glucagon, adrenocorticotrophic hormone (ACTH), corticotropin-releasing hormone (CRH), secretin, growth hormone, insulin, growth hormone-releasing hormone (GRH), vasopressin, oxytocin, gastrin, glucagon-like peptide (GLP-1, GLP-2, 7-36 amide), cholecystokinin, vasoactive intestinal polypeptide (VIP), pituitary adenolate cyclase activating polypeptide (p.a.c.a.p.), gastrin releasing hormone, galanin, thyroid-stimulating hormone (TSH), luteinizing hormone-releasing hormone (LH-RH), calcitonin, parathyroid hormone (PTH, PTH(1-34), PTH(1-84), peptide histidine isoleucine (PHI), neuropeptide Y (nP.Y)), peptide YY (P.YY), pancreatic polypeptide (P.P.), somatostatin, TGF-.alpha., TGF-.beta., nerve growth factor, fibroblast growth factor, relaxin, prolactin, natriuretic peptide, angiotensin, and brain derived nutrient factor. 5. A process according to claim 4, wherein the natriuretic peptide selected from the group consisting of ANP, BNP and CNP. 6. A process according to claim 3, wherein the physiologically active polypeptide is an enzyme. 7. A process according to claim 6, wherein the enzyme is KEX2 endopeptidase. 8. A process according to claim 6, wherein the enzyme is a proteolytic enzyme. 9. A process according to claim 8, further comprising the steps of: (a) expressing the desired polypeptide as an inactive fusion protein in host cells; (b) disrupting said host cells; (c) separating the fusion protein; (d) solubilizing the fusion protein with a denaturating agent; and (e) cleaving the linker peptide region with a protease intrinsic to the host cells so as to obtain the desired polypeptide from the fusion protein. 10. A process according to claim 9, wherein the protease intrinsic to the host cells and the fusion protein exist in the same fractions during an isolation process after the cell disruption. 11. A process according to claim 9, wherein the step (e) is carried out by decreasing a concentration of the denaturating agent. 12. A process according to claim 1, wherein the protease intrinsic to the host cells is E. coli ompT protease. 13. A process according to claim 9, wherein the denaturating agent for solubilization of the fusion protein is selected from the group consisting of urea, guanidine hydrochloride and surfactants. 14. A process according to claim 13, wherein the denaturating agent is 1 to 6 M urea. 15. A process according to claim 9, wherein the protease intrinsic to the host cells is E. coli ompT protease, and the fusion protein is cleaved with said protease in a solution containing 1 to 6 M urea. 16. A process according to claim 2, wherein the linker peptide has a site specifically recognized by a protease intrinsic to the host cells comprising an expression vector for the desired polypeptide. 17. A process according to claim 2, wherein the linker peptide consists of 2 to 50 amino acid residues and contains 1 or 2 pairs of basic amino acids. 18. A process according to claim 17, wherein the linker peptide has the basic amino acid pairs at the N-terminal and the C-terminal of the linker peptide. 19. A process according to claim 17, wherein the linker peptide has the amino acid sequence RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1). 20. A process according to claim 1, wherein the desired polypeptide has 20 to 800 amino acid residues. 21. A process according to claim 8, wherein the proteolytic enzyme is Staphylococcus aureus V8 protease or a derivative thereof having the amino acid sequence of SEQ ID No. 7 from amino acid 125 to amino acid 336 and having protease activity. 22. A process according to claim 8, wherein the proteolytic enzyme is Staphylococcus aureus V8 protease or a derivative thereof having protease activity and having the amino acid sequence selected from the group consisted of SEQ ID NO:5 from amino acid 125 to amino acid 344, SEQ ID NO:6 from amino acid 125 to amino acid 392, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24. 23. A process according to claim 1, wherein the protective polypeptide is a polypeptide derived from E. coli .beta.-galactosidase and/or polypeptide derived from aminoglycoside 3'-phosphotransferase of transposone 903 origin. 24. A process according to claim 2, wherein the protective polypeptide A is a polypeptide derived from E. coli .beta.-galactosidase, and the protective polypeptide C is a polypeptide derived from aminoglicoside 3'-phosphotransferase of transposone 903 origin. 25. A process according to claim 12, wherein the desired polypeptide excised by ompT protease in the presence of a denaturating agent is refolded by decreasing the concentration of the denaturating agent to obtain an active desired polypeptide. 26. A process according to claim 25, wherein the desired polypeptide is a derivative of Staphylococcus aureus having the amino acid sequence of SEQ ID No. 7 from amino acid 125 to amino acid 336 and having protease activity. 27. A process for the production of a desired polypeptide, comprising the steps of: (1) transforming Escherichia coli host cells with an expression vector comprising a gene coding for a fusion protein comprising at least one protective polypeptide, a desired polypeptide and a linker peptide, wherein the protective polypeptide is a polypeptide derived from E. coli .beta.-galactosidase and/or a polypeptide derived from an aminoglycoside 3'-phosphotransferase of transposone 903 origin, the desired polypeptide is a derivative of Staphylococcus aureus V8 protease, wherein the derivative has the amino acid sequence of SEQ ID No. 7 from amino acid 125 to amino acid 336 and has protease activity, the linker peptide between said protective polypeptide and said desired polypeptide has a substrate site specifically recognized by a protease intrinsic to the host cells; (2) expressing said gene in E. coli host cells to produce the derivative of the Staphylococcus aureus V8 protease as an inactive insoluble fusion protein, which fusion protein forms inclusion bodies wherein said inclusion bodies further comprise E. coli ompT protease which is a protease intrinsic to the host cells; (3) disrupting the cells so as to separate the fusion protein, and obtaining a fraction containing the E. coli ompT protease and the fusion protein; (4) solubilizing the fusion protein with a denaturating agent; and (5) decreasing a concentration of the denaturating agent to a level at which the E. coli ompT protease exhibits its activity to cleave the linker peptide with the protease so as to obtain the desired polypeptide from the fusion protein. 28. A process according to claim 27, wherein the denaturating agent for solubilizing the fusion protein is selected from the group consisting of urea, guanidine hydrochloride and a surfactant. 29. A process according to claim 28, wherein a concentration of the urea is 1 to 8 M. 30. A process according to claim 29, wherein a concentration of urea during the cleavage of the fusion protein with the E. coli ompT protease is about 4 M. 31. A process according to claim 27, wherein the linker peptide consists of 2 to 50 amino acid residues and contains pairs of two basic amino acids at the N- and C-terminal of the linker peptide or in the linker peptide. 32. A process according to claim 27, wherein the linker peptide has the amino acid sequence RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1). 33. A process according to claim 27, wherein the desired polypeptide is a derivative of the Staphylococcus aureus V8 protease having protease activity and having the amino acid sequence selected from the group consisting of SEQ ID NO:5 from amino acid 125 to amino acid 344, SEQ ID NO:6 from amino acid 125 to amino acid 392, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24. 34. A process according to claim 27, further comprising the step of (6) refolding the desired polypeptide obtained in the step (5) to obtain an active form of the desired polypeptide.

Details for Patent 6,037,145

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2014-09-07