Process for the production of naturally folded and secreted proteins by co-secretion of molecular chaperones

• US 6,455,279 B1
• Filed: 07/19/2000
• Issued: 09/24/2002
• Est. Priority Date: 07/29/1999
• Status: Expired due to Term

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Ambrosius, Dorothee, Schaeffner, Joerg, Schwarz, Elisabeth, Rudolph, Rainer
• Primary Examiner(s): Campbell, Eggerton A.
• Assistant Examiner(s): CHUNDURU, SURYAPRABHA
• Application Number:
  US09/618,869
• Time in Patent Office: 797 Days
• Field of Search: 435/6, 435/69.1, 435/71.1, 530/402
• US Class Current: 435/69.1
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 9/6459 t-plasminogen activator (3.4.21.68), i.e. tPA
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 304/21069 Protein C activated (3.4.21.69)

Abstract

A process for the production of a naturally folded eukaryotic polypeptide containing two or several cysteines linked by disulfide bridges by a) culturing prokaryotic cells in which the said prokaryotic cells contain an expression vector which codes for the said polypeptide which contains a prokaryotic signal sequence at the N-terminus, b) secreting the polypeptide into the periplasm or the medium, c) cleaving the signal sequence and isolating the polypeptide from the periplasm or the medium, which is characterized in that a nucleic acid coding for a molecular chaperone is additionally expressed in the said prokaryotic cell and the chaperone is secreted into the periplasm, is suitable for the recombinant production of polypeptides in prokaryotes in a high yield.

Claims

1. A process for the production of a naturally-folded eukaryotic polypeptide containing at least two cysteines linked by disulfide bridges, which comprises

a) culturing in a nutrient medium prokaryotic cells which contain (i) an expression vector that encodes the polypeptide, and contains a prokaryotic signal sequence at its N-terminus, and (ii) an expression vector that encodes a molecular chaperone naturally occurring in the cytoplasm of the prokaryotic cells, the culturing being under conditions such that the polypeptide and the chaperone is secreted into the periplasm of the prokaryotic cells or into the medium,

b) cleaving the signal sequence from the polypeptide;

and

c) isolating the polypeptide.

2. The process as claimed in claim 1, wherein the chaperone is a small heat shock protein (sHsp type) or a heat shock protein with a molar mass of about 40 kDa (Hsp40 type).

3. The process as claimed in claim 2, wherein a reducing thiol reagent is added to the nutrient medium.

4. The process as claimed in claim 3, wherein glutathione (GSH) is the reducing thiol reagent.

5. The process as claimed in claim 4, wherein the signal sequence is derived from gram-negative bacteria.

6. The process as claimed in claim 5, wherein the vector encoding the molecular chaperone and the vector encoding the polypeptide are two separate vectors.

7. The process as claimed in claim 5, wherein the vector encoding the molecular chaperone and the vector encoding the polypeptide are the same vector.

8. The process as claimed in claim 6, wherein vector encoding the molecular chaperone contains recombinant DNA encoding the molecular chaperone in operative linkage with DNA encoding a signal peptide for penetrating the inner bacterial membrane.

9. The process as claimed in claim 7, wherein vector contains recombinant DNA encoding the molecular chaperone in operative linkage with DNA encoding a signal peptide for penetrating the inner bacterial membrane.

10. The process as claimed in claim 8, wherein the DNA encoding the secreted molecular chaperone and/or for the secreted protein is under the control of an inducible expression signal.

11. The process as claimed in claim 8, wherein the DNA encoding the secreted molecular chaperone is under the control of an inducible expression signal.

12. The process as claimed in claim 8, wherein the DNA encoding the secreted protein is under the control of an inducible expression signal.

13. The process as claimed in claim 9, wherein the DNA encoding the secreted molecular chaperone is under the control of an inducible expression signal.

14. The process as claimed in claim 9, wherein the DNA encoding the secreted protein is under the control of an inducible expression signal.

15. The process as claimed in claim 8, wherein the polypeptide is an antibody, antibody fragment, interferon, protein hormone or a protease.

16. The process as claimed in claim 8, wherein the polypeptide is an antibody, antibody fragment, interferon, protein hormone or a protease.