Method for improved protein expression in bacteria by monitoring and modulating protein folding

• US 20040185528A1
• Filed: 01/05/2004
• Published: 09/23/2004
• Est. Priority Date: 07/06/2001
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Horn, Uwe, Riesenberg, Ulrike, Strittmatter, Wolfgang
• Granted Patent: US 7,943,300 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

This invention relates to a new method for improving functional protein expression whereby the folding process is monitored by online measurement and, if required, the protein folding is influenced by adding folding promoting agents and/or co-expression of the periplasmic chaperone (Skp). In this respect, the invention offers a technology to improve the yield of functionally expressed recombinant proteins.

Claims

1. A method for monitoring functionally correct folding or misfolding of a target protein in vivo in a recombinant process comprising applying a reporter gene, that is under the control of the promoter of the periplasmatic protease DegP, to an expression system expressing said target protein, wherein the production of the gene product of the reporter gene in said expression system is a measure for the incorrect folding of the target protein.

2. A method according to claim 1, wherein the DegP promoter sequence is modified close to the start codon of the reporter gene which is fused to the promotor in downstream direction.

3. A method according to claim 2, wherein said DegP promoter has the sequence as indicated in FIG. 1.

4. A method according to any of the claims 1 to 3, wherein the reporter gene is the luciferase gene (luc⁺).

5. A method according to claim 4, wherein a DegP-luc⁺ gene cassette cloned into a vector is used.

6. A method of claim 5, wherein said vector is plt1 comprising the DNA sequences as specified in FIG. 7.

7. A method according to any of the claims 1 to 6, wherein the production of the reporter gene product is monitored by an online kinetic measurement process.

8. A method for optimizing functionally correct folding of a target protein in an expression and fermentation process comprising (i) monitoring the folding of said target protein according to a method of any of the claims 1 to 7, (ii) modulating protein folding by co-expressing of the periplasmatic chaperone Skp and / or adding one or more agents that promote functionally correct protein folding, and optionally (iii) repeating step (i) and (ii) until an optimum of functional-correctly folded protein is obtained.

9. A method of claim 8, wherein said folding promoting agent is a polyole and/or a betaine derivative.

10. A method of claim 9, wherein said folding promoting agent is selcted from the group consisting of glycerol, sorbitol, glycine betaine, hydroxyectoine.

11. A method according to any of the claims 8 to 10, wherein a bacterial expression system is used.

12. A method of claim 11, wherein said bacterial expression system is E. coli.

13. A method of claim 12, wherein said E-coli is RV308 (ATCC 31608).

14. A method according to any of the claims 8 to 13, wherein a dual plasmid system is used consisting of a first plasmid comprising the promoter/reporter genes and second plasmid comprising the gene coding for the target protein the folding of which shall be optimized when expressed.

15. A method according to any of the claims 8 to 14, wherein monitoring and modulating are carried out online and simultaneously during the production phase.

16. A method for producing a recombinant target protein having optimized folding properties by culturing bacterial host cells in a nutrient and isolating and purifying said optimized folded target protein, said method comprising a method of any of the claims 8 to 5.

17. A method of claim 16, wherein said target protein is a fusion protein.

18. A method of claim 17, wherein said fusion protein is a single-chain Fv and/or mini antibody.

19. A kit for expressing a target protein having improved folding properties in a bacterial host cell system consisting of a first package comprising a plasmid containing the gene of said target protein, a second package comprising a plasmid containing the DegP-luc+ gene cassette, and a third package containing the bacterial host cells, which are transformed with said plasmids.