Claims for Patent: 6,103,495

Summary for Patent: 6,103,495

Title:	Direct expression of peptides into culture media
Abstract:	Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with novel vectors, a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special vectors are provided which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.
Inventor(s):	Mehta; Nozer M. (Randolph, NJ), Consalvo; Angelo P. (Monroe, NY), Ray; Martha V. L. (Nutley, NJ), Meenan; Christopher P. (Lincoln Park, NJ)
Assignee:	Unigene Laboratories, Inc. (Fairfield, NJ)
Application Number:	09/060,765
Patent Claims:	1. An expression vector comprising a plurality of transcription cassettes, each cassette comprises: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac. 2. The vector of claim 1, wherein said control region has exactly two promoters. 3. The vector of claim 1, wherein said tac promoter is 5' of another promoter in said control region. 4. The vector of claim 1, wherein the control region comprises both a tac promoter and a lac promoter. 5. The vector of claim 4, wherein the lac promoter is 3' of the tac promoter. 6. The vector of claim 1, wherein said nucleic acids coding for the signal peptide encode a signal peptide for secreted bacterial proteins. 7. The vector of claim 6, wherein said signal is OmpA signal peptide. 8. The vector of claim 1, wherein said peptide product has a molecular weight of less than 10 KDa. 9. The vector of claim 1, wherein the C-terminal amino acid of said peptide product is glycine. 10. The vector of claim 9, wherein said peptide product is salmon calcitonin precursor. 11. The vector of claim 9, wherein said peptide product is calcitonin gene related peptide precursor. 12. An expression vector comprising: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac, wherein the C-terminal amino acid of said peptide product is glycine and said peptide product is parathyroid hormone. 13. A host cell transformed or transfected with the vector of claim 1. 14. The host cell of claim 13, wherein said host cell is a bacterial cell. 15. The host cell of claim 14, wherein said bacterial cell is a gram negative bacterial cell. 16. The host cell of claim 14, wherein said bacterial cell is E. coli. 17. A host cell transformed or transfected with an expression vector which comprises: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac, wherein said host cell is a BLR E. coli bacterial strain. 18. A host cell transformed or transfected with an expression vector which comprises: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding reuion, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac, wherein said host cell is a BL21 E. coli bacterial strain. 19. The host cell of claim 16, wherein said E. coli is strain WA837. 20. An E. coli host containing and expressing an expression vector which comprises a plurality of transcription cassettes in tandem, each cassette comprising: (a) a coding region comprising nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters in tandem and at least one ribosome binding site, wherein at least one of said promoters is selected from the group consisting of tac and lac. 21. The host of claim 20, wherein said control region comprises from 5' to 3' a tac promoter and a lac promoter. 22. The host of claim 20, wherein said vector has exactly two transcription cassettes in tandem. 23. A host cell transformed with an expression vector which comprises a gene for expressing salmon calcitonin precursor, said host cell being E. coli strain BLR. 24. A host cell transformed with an expression vector which comprises a gene for expressing calcitonin gene related peptide precursor, said host being E. coli strain BLR. 25. A method of producing a peptide product which comprises culturing the host cell of claim 13 in a culture medium and then recovering the peptide product from the medium in which the host cell has been cultured. 26. A method of producing a peptide product which comprises culturing a host cell transformed or transfected with an expression vector in a culture medium and then recovering the peptide product from the medium in which the host cell has been cultured, wherein: peptide product yield exceeds 100 mg per liter of media, and said expression vector comprises: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac. 27. A method of producing salmon calcitonin precursor which comprises culturing a host cell in a culture medium and then recovering the salmon calcitonin precursor from the medium in which the host cell has been cultured, wherein said host cell is E. coli strain BLR transformed with an expression vector which comprises a gene for expressing salmon calcitonin precursor. 28. The method of claim 25, wherein the peptide product is salmon calcitonin precursor. 29. The method of claim 25, wherein the peptide product is calcitonin gene related peptide precursor. 30. An expression vector comprising: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac, wherein said peptide product is parathyroid hormone. 31. A method of producing a peptide product which comprises culturing a host cell transformed or transfected with an expression vector in a culture medium and then recovering the peptide product from the medium in which the host cell has been cultured, wherein: said peptide product is parathyroid hormone; and said expression vector comprises: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac. 32. A method of producing a peptide product which comprises culturing a host cell transformed or transfected with an expression vector in a culture medium and then recovering the peptide product from the medium in which the host cell has been cultured, wherein: said expression vector comprises: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac, and wherein recovering said peptide product comprises: (a) separating host cells from the culture medium; and (b) subjecting the medium to reverse-phase liquid chromatography and recovering fractions containing peptide product; (c) subjecting said fractions of step (b) to cation exchange chromatography; and (d) thereafter recovering fractions containing peptide product. 33. A method of producing a peptide product which comprises culturing a host cell transformed or transfected with an expression vector in a culture medium and then recovering the peptide product from the medium in which the host cell has been cultured, wherein: said expression vector comprises: (a) a coding region with nucleic acids coding for a peptide product coupled in reading frame 3' of nucleic acids coding for a signal peptide; and (b) a control region linked operably with the coding region, said control region comprising a plurality of promoters and at least one ribosome binding site, wherein at least one of said promoters is tac, and wherein recovering said peptide product comprises: (a) separating host cells from the culture medium; (b) subjecting the medium to cation exchange chromatography and recovering fractions containing said peptide product; and (c) subjecting the recovered fraction of step (b) to reverse-phase liquid chromatography and recovering fractions containing peptide product; (d) subjecting the recovered fractions of step (c) to cation exchange chromatography; and (e) thereafter recovering fractions containing peptide product.

Details for Patent 6,103,495

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2017-04-16