Methods for production of proteins
View Patent ↗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.
1. A method for making a stained molecular weight ladder, said method comprising:
(a) producing a plurality of polypeptides of different molecular weights comprising thioredoxin or a thioredoxin deletion mutant
(b) incubating the plurality of polypeptides with at least one protein-binding dye to form a plurality of pre-stained molecular weight markers; and
(c) admixing the plurality of pre-stained markers to form a pre-stained molecular weight ladder.
2. The method of claim 1 , further comprising admixing the plurality of polypeptides of different molecular weights comprising thioredoxin or a thioredoxin deletion mutant with one or more additional stained polypeptides to form a set of pre-stained molecular weight standards.
3. The method of claim 1 , wherein the producing step comprises producing a plurality of polypeptides of different molecular weights comprising thioredoxin or a truncated thioredoxin.
4. The method of claim 1 , wherein the producing step comprises producing a plurality of polypeptides of different molecular weights comprising thioredoxin or a thioredoxin deletion mutant having the ability to form inclusion bodies upon expression in a bacterial host cell.
5. The method of claim 3 , wherein the producing step comprises producing a plurality of polypeptides of different molecular weights comprising thioredoxin or a truncated thioredoxin having the ability to form inclusion bodies upon expression in a bacterial host cell.
6. The method of claim 5 , wherein the producing step comprises producing a polypeptide comprising a carboxy terminal-truncated thioredoxin having the ability to form inclusion bodies upon expression in a bacterial host cell.
7. The method of claim 5 , wherein the producing step comprises producing a polypeptide comprising a thioredoxin having a truncation of between 2 and 50 carboxy terminal amino acids.
8. The method of claim 5 , wherein the producing step comprises producing a polypeptide comprising a thioredoxin having a truncation of between 33 and 50 carboxy terminal amino acids.
9. The method of claim 5 , wherein the producing step comprises producing a polypeptide comprising a thioredoxin having a truncation of between 2 and 33 carboxy terminal amino acids.
10. The method of claim 5 , wherein the producing step comprises producing a polypeptide comprising a thioredoxin having a truncation of between 2 and 22 carboxy terminal amino acids.
11. The method of claim 5 , wherein the producing step comprises producing a polypeptide comprising a thioredoxin having a truncation of between 23 and 33 carboxy terminal amino acids.
12. The method of claim 5 , wherein the producing step comprises producing a polypeptide comprising a thioredoxin having a truncation of 23 carboxy terminal amino acids.
13. The method of claim 1 , wherein the producing step comprises producing a polypeptide comprising a thioredoxin having a molecular weight of 10 kD.
14. The method of claim 3 , wherein the producing step comprises producing a polypeptide comprising a carboxy terminal-truncated form of Escherichia coli thioredoxin, wherein the carboxy truncated form of Escherichia coli thioredoxin is encoded by a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:8.
15. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights comprises polypeptides having molecular weight increments of 5 kD, 10 kD, 20 kD, 25 kD, 50 kD, or 100 kD.
16. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights comprises polypeptides having molecular weight increments of 5 kD.
17. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights comprises polypeptides having molecular weight increments of 10 kD.
18. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights range from 5 kD to 300 kD.
19. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights range from 5 kD to 250 kD.
20. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights range from 10 kD to 220 kD.
21. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights comprises at least one recombinant fusion protein that comprises thioredoxin or a thioredoxin deletion mutant fused to at least one other polypeptide.
22. The method of claim 1 , wherein the plurality of polypeptides of different molecular weights comprises at least one recombinant fusion protein that comprises multimers of thioredoxin or a thioredoxin deletion mutant.