Recombinant protein expression

• US 20050064545A1
• Filed: 01/07/2003
• Published: 03/24/2005
• Est. Priority Date: 01/07/2002
• Status: Abandoned Application

• Current Assignee: European Molecular Biology Laboratory
• Original Assignee: European Molecular Biology Laboratory
• Inventors: Deuerling, Elke, DeMarco, Ario, Geerlof, Arie, Bukau, Bernd
• Application Number:
  US10/500,883
• Publication Number:
  US 20050064545A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

There are provided methods for the expression of a recombinant protein of interest, said methods comprising, in additional to various additional steps: a) culturing a host cell which expresses: i) one or more genes encoding the recombinant protein(s) of interest; ii) at least two genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GRoES, Dnak, DnaJ, GRpe, ClpB and their homologs (for example, Hsp104, Ydj1 and Ssa1 in yeast); under conditions suitable for protein expression; and separating said recombinant protein of interest from the host cell culture. Also provided are methods for increasing the degree of refolding of a recombinant protein of interest by ading a composition containing a chaperone protein to a preparation of the recombinant protein of interest in vitro.

Claims

1. A method for the expression of a recombinant protein of interest, said method comprising:

a) culturing a host cell which expresses;

i) one or more genes encoding the recombinant protein(s) of interest;

ii) at least two genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs under conditions suitable for protein expression; and

b) separating said recombinant protein of interest from the host cell culture.

2. A method according to claim 1, wherein the genes selected in step a) ii) include DnaK, DnaJ and GrpE or homologs thereof.

3. A method according to claim 2, wherein the genes selected in step a) ii) additionally include ClpB or a homolog thereof.

4. A method according to claim 1, wherein the genes selected in step a) ii) include GroES and GroEL or homologs thereof.

5. A method according to claim 4, wherein the genes selected in step a) ii) include the DnaK, DnaJ, GrpE, ClpB, GroES and GroEL genes or homologs thereof.

7. A method according to claim 1 wherein the levels of the respective chaperone proteins are controlled.

8. A method according to claim 7, wherein said levels of chaperone proteins are controlled by expressing the genes encoding the respective chaperone proteins from different promoters.

9. A method according to claim 7, wherein the respective chaperone proteins are expressed using expression systems of different strength.

10. A method according to claim 7, wherein said chaperone proteins are over-expressed relative to the expression levels that occur naturally in non-recombinant cells.

11. A method according to claim 1, wherein the levels of the chaperone proteins relative to the recombinant protein(s) of interest are controlled by expressing the genes encoding the respective proteins from different promoters or by using different polymerases.

12. A method according to claim 1, wherein in culturing step a) of the method, a block in protein synthesis is imposed, for example, by the addition of an effective amount of a protein synthesis inhibitor to the culture system, once a desired level of recombinant protein of interest has accumulated.

13. A method according to claim 12, wherein the chosen protein synthesis inhibitor is chloramphenicol, tetracycline, gentamycin or streptomycin.

14. A method according to claim 1, wherein in culturing step a) of the method, a reduction in gene transcription is imposed, for example, by removal of any agents that are effective to induce recombinant protein expression, or via the addition of a transcription blocking compound, once a desired level of recombinant protein of interest has accumulated

18. A method according claim 14, wherein in step a) ii), a combination of chaperone proteins is expressed.

20. A method according to claim 1, wherein said cultured host cell is a prokaryotic cell, such as an E. coli cell, a Lactococcus cell, a Lactobacillus cell or a Bacillus subtilis cell, or a eukaryotic cell such as a yeast cell, for example a Pichia or Saccharomyces yeast cell, or an insect cell, for example after baculoviral infection.

21. A method according to claim 1, wherein an optimised yield of said recombinant protein of interest is manifested by increasing the level of de novo protein folding.

22. A method according to claim 1, wherein an optimised yield of said recombinant protein of interest is manifested by increasing the level of in vivo refolding of aggregated, or misfolded soluble, recombinant protein.

23. A method according to claim 1, wherein an optimised yield of said recombinant protein of interest is manifested by increasing the level of in vitro refolding of aggregated, or misfolded soluble, recombinant protein.

24. A method according to claim 20, wherein an optimised yield of said recombinant protein is manifested by increasing the level of de novo protein folding in combination with an increased level of in vivo protein refolding and/or in vitro protein refolding.

25. A method according to claim 21, wherein said increased level of folding or re-folding results in increased solubility of the recombinant protein of interest.

26. A method according to claim 21, wherein said increased level of folding or re-folding results in increased activity of the recombinant protein of interest.

34. A method according claim 1, wherein the refolding temperature and time course of refolding are controlled.

41. A method according to claim 2, wherein the genes selected in step a) ii) include GroES and GroEL or homologs thereof

42. A method according to claim 3, wherein the genes selected in step a) ii) include GroES and GroEL or homologs thereof.

43. A method according to claim 41, wherein the genes selected in step a) ii) include the DnaK, DnaJ, GrpE, ClpB, GroES and GroEL genes or homologs thereof.

44. A method according to claim 42, wherein the genes selected in step a) ii) include the DnaK, DnaJ, GrpE, ClpB, GroES and GroEL genes or homologs thereof.

6. A method for the expression of a recombinant protein of interest, said method comprising:

a) culturing under conditions suitable for protein expression a host cell which expresses;

i) one or more genes encoding one or more recombinant protein(s) of interest;

ii) one or more genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs;

iii) one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family small heatshock proteins of the IbpB family and homologs thereof; and

(b) separating said recombinant protein of interest from the host cell culture.

15. A method for the expression of a recombinant protein of interest, said method comprising:

a) culturing a host cell which expresses;

i) one or more genes encoding the recombinant protein(s) of interest;

ii) one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs;

under conditions suitable for protein expression;

b) imposing a block in protein synthesis, for example, by the addition of an effective amount of a protein synthesis inhibitor to the culture system, once a desired level of recombinant protein of interest has accumulated; and

c) separating said recombinant protein of interest from the host cell culture.

17. A method according to claim 15, wherein said host cells additionally expresses one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family and/or the IbpB family and/or their homologs.

19. A method according to claim 15, wherein the chosen protein synthesis inhibitor is chloramphenicol, tetracycline, gentamycin or streptomycin.

16. A method for the expression of a recombinant protein of interest, said method comprising:

a) culturing a host cell which expresses;

i) one or more genes encoding the recombinant protein(s) of interest;

ii) one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs;

under conditions suitable for protein expression;

b) imposing a reduction in gene transcription, for example, by removal of any agents that are effective to induce recombinant protein expression, or via the addition of a transcription blocking compound, once a desired level of recombinant protein of interest has accumulated; and

c) separating said recombinant protein of interest from the host cell culture.

45. A method according to claim 16, wherein said host cells additionally expresses one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof.

27. A method for increasing the degree of refolding of a recombinant protein of interest, said method comprising adding a composition containing a chaperone protein to a preparation of the recombinant protein of interest in vitro.

28. A method according to claim 27, wherein a combination of chaperone proteins is added to the preparation of the recombinant protein of interest.

29. A method according to claim 27, wherein the preparation of the recombinant protein of interest is a preparation of soluble recombinant protein that has been precipitated in vivo.

30. A method according to claim 27, wherein the preparation of the soluble recombinant protein of interest is a preparation of in vitro precipitated recombinant protein.

31. A method according claim 27, wherein said composition containing the chaperone protein(s) is added after removal of any agents that are effective to induce soluble recombinant protein expression or after addition of a transcription blocking compound.

32. A method according to claim 27, additionally comprising the step of imposing a block in protein synthesis, such as by the addition of an effective amount of a protein synthesis inhibitor to the culture system.

33. A method according to claim 32, wherein the chosen protein synthesis inhibitor is chloramphenicol, tetracycline, gentamycin or streptomycin.

35. A method according to claim 27, additionally comprising the use of one or more proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the heatshock proteins of the IbpB family and homologs thereof.

36. The use of one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof, in the manufacture of a medicament for the treatment of disease in which the presence of aggregated proteins are implicated.

37. The use of one or more selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof, in the manufacture of a medicament for the treatment of disease in which the presence of aggregated proteins are implicated.

38. A method of treating a patient suffering from a disease in which the presence of aggregated proteins is implicated, comprising administering one or more genes encoding one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof.

40. The method of claim 38, wherein the disease is late or early onset Alzheimer'"'"'s disease, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, or a spongiform encephalopathy.

39. A method of treating a patient suffering from a disease in which the presence of aggregated proteins is implicated, comprising administering one or more proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and their homologs, and one or more proteins selected from the group consisting of the small heatshock proteins of the IbpA family, the small heatshock proteins of the IbpB family and homologs thereof.

46. The method of claim 39, wherein the disease is late or early onset Alzheimer'"'"'s disease, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, or a spongiform encephalopathy.