Protein production
A method for producing from host cells a heterologous polypeptide or protein comprising at least one cysteine residue in a reduced state. The method cultured the host cells in a culture medium comprising a reducing agent; and recovered the heterologous polypeptide or protein comprising at least one cysteine residue in a reduced state from the host cells or from the culture medium, where the host cells comprise a nucleic acid encoding the heterologous protein or polypeptide, and where the reducing agent is capable of permeating the plasma membrane of the host cells.
1. A method for producing from host cells an active heterologous polypeptide or protein comprising at least one cysteine residue in a reduced state, the method comprising
(i) culturing host cells in a culture growth medium comprising a reducing agent having a reduction potential stronger than −0.1V at pH 7 and 25° C.; and
(ii) recovering the active heterologous polypeptide or protein comprising at least one cysteine residue in a reduced state from said host cells or from the culture growth medium, where the host cells comprise a nucleic acid encoding the heterologous protein or polypeptide, and where the reducing agent is capable of permeating the plasma membrane of the host cells.
2. The method of claim 1 where the host cells are prokaryotic cells.
3. The method of claim 2 where the prokaryotic cells are bacterial cells.
4. The method of claim 3 where the bacterial cells are Escherichia coli cells.
5. The method of claim 1 where the heterologous polypeptide or protein comprises at least two cysteine residues in a reduced state.
6. The method of claim 1 where a native conformation of the heterologous polypeptide or protein comprises at least one cysteine residue.
7. The method of claim 1 where oxidation of the at least one cysteine residue reduces activity of the produced heterologous polypeptide or protein.
8. The method of claim 1 where the heterologous polypeptide or protein is a ribonuclease inhibitor.
9. The method of claim 1 where the reducing agent is a thiol containing compound.
10. The method of claim 9 where the reducing agent comprises at least two thiol groups.
11. The method of claim 1 where the reducing agent has a reduction potential stronger than −0.2V at pH 7 and 25° C.
12. The method of claim 1 where the reducing agent is dithiothreitol or dithioerythritol.
13. The method of claim 12 where the reducing agent is present in the culture growth medium at a concentration of 2 mM to 18 mM.
14. The method of claim 1 where the heterologous polypeptide or protein is recovered from the cytoplasm of the host cells.
15. The method of claim 1 where the host cell is a prokaryotic cell and the heterologous polypeptide or protein is recovered from the periplasm of the prokaryotic cell.
16. The method of claim 1 where step (i) is conducted at a temperature below 37° C.
17. The method of claim 16 where the temperature is between 15° C. and 30° C.
18. The method of claim 1 where during step (i) the concentration of the oxygen in the medium is less than 0.8%.
19. The method of claim 1 where the nucleic acid encoding the heterologous protein or polypeptide is expressed under the control of a pCU promoter, and/or comprises -var or -lac ribosome binding sites.
20. The method of claim 1 wherein the host cells further express molecular chaperones.
21. The method of claim 20 where the molecular chaperones are GroEL and GroES.