Methods of production of proteins

• US 20060269992A1
• Filed: 01/11/2006
• Published: 11/30/2006
• Est. Priority Date: 01/08/1997
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Chatterjee, Deb K., Flynn, Elizabeth, Longo, Mary, Oberfelder, Robert
• Granted Patent: US 7,265,206 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 1/26 Electrophoresis
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C12N 9/0036 acting on NADH or NADPH (1.6)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The current invention provides methods for producing a polypeptide as inclusion bodies in bacterial host cells. The present methods are carried out by forming a gene construct comprising the genetic sequence encoding a polypeptide operatively linked to that of an inclusion partner protein, such as E. coli thioredoxin or a modified E. coli thioredoxin, such that host cells comprising the gene construct produce the polypeptide as intracellular inclusion bodies. The methods of the present invention facilitate the rapid isolation and purification of recombinant proteins. In addition, the present methods may be useful for producing polypeptides or proteins which are small and are typically difficult to express, as well as those proteins that are toxic to host cells such as E. coli. The present invention also provides plasmids, vectors and host cells to be used in the present invention for production of polypeptides, and methods of production of polypeptides using these vectors and host cells. The invention further provides methods for producing protein molecular weight ladders for use in protein gel electrophoresis, as well as proteins and protein molecular weight ladders produced by these methods.

Claims

1. (canceled)

2. A method for making a stained molecular weight marker, the method comprising:

(1) producing a first thioredoxin polypeptide comprising thioredoxin, a modified thioredoxin, or a truncated thioredoxin; and

(2) incubating the first thioredoxin polypeptide with a protein-binding dye to form a first stained molecular weight marker.

3. The method of claim 2, wherein the incubating step is incubating the first thioredoxin polypeptide with a protein-binding dye to form a first stained molecular weight marker that has a molecular weight of between 5 kD and 330 kD.

4. The method of claim 2, further comprising:

(3) admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a plurality of pre-stained molecular weight markers.

5. The method of claim 4, wherein the admixing step is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a plurality of pre-stained molecular weight markers having molecular weights of between 5 kD and 330 kD.

6. The method of claim 4, wherein the admixing step is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a plurality of pre-stained molecular weight markers having a molecular weights between 10 kD and 250 kD.

7. The method of claim 4, wherein the admixing step is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a pre-stained molecular weight ladder that comprises from 3-20 proteins of different sizes.

8. The method of claim 4, wherein the admixing step is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a pre-stained molecular weight ladder comprising at least one protein of a molecular weight of from 10 kD to 50 kD.

9. The method of claim 4, wherein the admixing step is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a pre-stained molecular weight ladder comprising at least one protein of a molecular weight of from 50 kD to 100 kD.

10. The method of claim 4, wherein the admixing step is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a pre-stained molecular weight ladder that comprises at least one protein of a molecular weight of from about 100 kD to about 220 kD.

11. The method of claim 4, wherein admixing is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a pre-stained molecular weight ladder that comprises two or more proteins having molecular weight increments of 5 kD, 10 kD, 20 kD, 25 kD, 50 kD, or 100 kD.

12. The method of claim 4, wherein admixing is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a pre-stained molecular weight ladder that comprises two or more proteins having molecular weight increments of 10 kD.

13. The method of claim 4, wherein admixing is admixing the thioredoxin polypeptide with a plurality of stained polypeptides of different molecular weights to form a pre-stained molecular weight ladder that comprises at least three proteins having molecular weight increments of 10 kD or a multiple thereof.

14. The method of claim 2, wherein the producing step comprises producing a first thioredoxin polypeptide comprising an E. coli thioredoxin.

15. The method of claim 2, wherein the producing step comprises producing a first thioredoxin polypeptide comprising a modified thioredoxin.

16. The method of claim 15, wherein the producing step comprises producing a first thioredoxin polypeptide comprising a modified thioredoxin having the ability to form inclusion bodies upon expression in a bacterial host cell.

17. The method of claim 2, wherein the producing step comprises producing a first thioredoxin polypeptide comprising a truncated thioredoxin.

18. The method of claim 17, wherein the producing step comprises producing a first thioredoxin polypeptide comprising a carboxy terminal-truncated thioredoxin.

19. The method of claim 17, wherein the producing step comprises producing a first thioredoxin polypeptide comprising a thioredoxin having a truncation of between 2 and 50 carboxy terminal amino acids.

20. The method of claim 17, wherein the producing step comprises producing a first thioredoxin polypeptide having a molecular weight of 10 kD.

21. The method of claim 17, wherein the producing step comprises producing a first thioredoxin polypeptide comprising a thioredoxin having a carboxy terminal-truncated form of Escherichia coli thioredoxin which is encoded by a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:

8

22. The method of claim 2, wherein the producing step results in production of a first thioredoxin polypeptide that comprises a multimer of thioredoxin, a modified thioredoxin, or a truncated thioredoxin.

23. The method of claim 22, wherein the producing step results in production of the first thioredoxin polypeptide, wherein the first thioredoxin polypeptide comprises a multimer of a truncated thioredoxin.

24. The method of claim 22, wherein the producing step results in production of a first thioredoxin polypeptide that has a molecular weight of between 20 kD and 250 kD.

25. The method of claim 2, wherein the producing step results in production of a first thioredoxin polypeptide that comprises a fusion of thioredoxin, a modified thioredoxin, or a truncated thioredoxin and another polypeptide.

26. The method of claim 25, wherein the producing step results in production of a first thioredoxin polypeptide that has a molecular weight of between 15 kD and 220 kD.

27. The method of claim 2, further comprising:

(3) producing a second thioredoxin polypeptide comprising thioredoxin, a modified thioredoxin, or a truncated thioredoxin; and

(4) incubating the second thioredoxin polypeptide with a protein-binding dye to form a second stained molecular weight marker.

28. The method of claim 27, wherein said second stained molecular weight marker has a molecular weight of between 5 kD and 300 kD.

29. The method of claim 27, wherein the producing step results in production of the second thioredoxin polypeptide, wherein the second thioredoxin polypeptide comprises a multimer of thioredoxin, a modified thioredoxin, or a truncated thioredoxin.

30. The method of claim 27, wherein the second polypeptide is admixed with the first polypeptide to form a molecular weight marker set.

31. The method of claim 30, wherein the pre-stained molecular weight marker set comprises proteins having molecular weights of between 5 kD and 330 kD.

32. The method of claim 30, wherein the pre-stained molecular weight marker set comprises proteins having molecular weights between 10 kD and 250 kD.

33. The method of claim 27, further comprising:

(5) producing a third thioredoxin polypeptide comprising thioredoxin or a modified thioredoxin; and

(6) incubating the third thioredoxin polypeptide with a protein-binding dye to form a third stained molecular weight marker.

34. The method of claim 33, wherein said third stained molecular weight marker has a molecular weight of between 5 kD and 330 kD.

35. The method of claim 33, wherein the producing step results in production of the third thioredoxin polypeptide that comprises a multimer of thioredoxin, a modified thioredoxin, or a truncated thioredoxin.

36. The method of claim 33, wherein the third polypeptide is admixed with the first polypeptide and the second polypeptide to form a molecular weight marker set.

37. The method of claim 36, wherein the pre-stained molecular weight marker set comprises proteins having molecular weights of between 5 kD and 330 kD.

38. The method of claim 36, wherein the pre-stained molecular weight marker set comprises proteins having molecular weights of between 10 kD and 250 kD.

39. A pre-stained molecular weight marker comprising thioredoxin, a modified thioredoxin, or a truncated thioredoxin.

40. The pre-stained molecular weight marker of claim 39, wherein the molecular weight marker has a molecular weight of between 5 kD and 330 kD.

41. A molecular weight marker set comprising a plurality of pre-stained molecular weight markers, wherein at least two of the stained molecular weight markers comprise thioredoxin, a modified thioredoxin, or a truncated thioredoxin.

42. The molecular weight marker set of claim 41, wherein the plurality of pre-stained molecular weight markers have molecular weights of between 5 kD and 330 kD.