Claims for Patent: 6,136,564

Summary for Patent: 6,136,564

Title:	Process for the production of peptides by way of streptavidin fusion proteins
Abstract:	The invention relates to a process for recombinant preparation of peptides by expression of a DNA in micro-organisms, which DNA codes for a fusion protein made of streptavidin and one of the said peptides. Streptavidin and the peptide are bound by a peptide sequence which can be cleaved by an endoproteinase. The process also includes isolation of the insoluble, inactive protein, solublisation of the inactive protein using a denaturant, dilution of the denaturant at a pH value of between 8.5 and 11 until cleaving of the fusion protein by an endoproteinase can take place, cleaving of the fusion protein, lowering of the pH value until streptavin and non-cleaved fusion protein precipitate, and cleaning of the desired peptide from the supernatant. Said process is particularly suitably for producing parathromone and urodilatin and fragments thereof.
Inventor(s):	Kopetzki; Erhard (Penzberg, DE)
Assignee:	Roche Diagnostics GmbH (Mannheim, DE)
Application Number:	09/068,738
Patent Claims:	1. A process for recombinantly producing a peptide, the process comprising expressing, in a microorganism, a DNA which codes for a fusion protein comprising a streptavidin and the peptide to be produced, wherein the streptavidin and the peptide are linked via a cleavage sequence which is cleavable by a factor Xa or thrombin endoproteinase, to produce an insoluble fusion protein; isolating the insoluble fusion protein; solubilizing the fusion protein in a solution comprising a denaturing agent and adjusting the solution to a pH value of between 8.5 and 11; cleaving the fusion protein via the cleavage sequence using a factor X or thrombin endoproteinase to produce streptavidin cleavage segments and peptide cleavage segments; precipitating the streptavidin cleavage segments and uncleaved fusion protein from the solution by lowering the pH value of the solution; and purifying the peptide from the solution supernatant. 2. The process of claim 1, wherein the microorganism is prokaryotic. 3. The process of claim 2, wherein the insoluble fusion protein is isolated in inactive form. 4. The process of claim 3, wherein the insoluble fusion protein is isolated in an inclusion body. 5. The process of claim 1, wherein, in said precipitating step, the pH value of the solution is lowered below 6. 6. The process of claim 1, wherein the peptide to be produced is a natriuretic peptide or a parathyroid hormone peptide. 7. The process of claim 1, wherein the peptide to be produced is a urodilatin peptide or a parathyroid hormone peptide. 8. The process of claim 7, wherein the peptide to be produced is selected from the group consisting of a urodilatin peptide of amino acids 95-126 (SEO ID NO:2), a urodilatin peptide of amino acids 99-126 (amino acids 5-32 of SEQ ID NO:2), a urodilatin peptide of amino acids 102-126 (amino acids 8-32 of SEO ID NO:2) and a parathyroid hormone peptide of amino acids 1-37 (SEO ID NO:16). 9. The process of claim 1, wherein the cleavage sequence contains 5-15 amino acids and a C-terminal cleavage site. 10. The process of claim 9, wherein the cleavage sequence further contains a linker sequence comprising a plurality of amino acids attached at the N-terminal of the cleavage sequence, wherein each amino acid of the plurality of amino acids is independently selected from the group consisting of Gly, Thr, Ser, Ala, Pro, Asp, Glu, Arg and Lys. 11. The process of claim 10, wherein the linker sequence comprises 2-8 amino acids each of which is independently selected from the group consisting of Asp and Glu. 12. The process of claim 1, wherein the microorganism is eukaryotic. 13. The process of claim 1, wherein the solution is brought to a pH value of between 8,5 and 11 using art aqueous buffer.

Details for Patent 6,136,564

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2015-11-16