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 disulfide bond, the method comprising
(i) culturing host cells in a culture medium comprising a reducing agent having a reduction potential stronger than −0.1V at pH 7 and 25° C., to produce the heterologous polypeptide or protein with at least two cysteine residues in a reduced state, wherein a concentration of oxygen in the medium is less than 0.8%;
(ii) recovering the heterologous polypeptide or protein from said host cells or from the culture medium; and
(iii) exposing the heterologous polypeptide or protein to conditions which allow the at least two cysteine residues to form the at least one disulfide bonds; 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 wherein step (iii) is conducted as part of step (ii) or step (iii) is conducted subsequently to step (ii).
6. The method of claim 1 where the heterologous polypeptide or protein is a deoxyribonuclease inhibitor.
7. The method of claim 1 wherein step (iii) comprises exposing the heterologous polypeptide or protein to calcium ions.
8. The method of claim 1 where the reducing agent is a thiol containing compound.
9. The method of claim 8 where the reducing agent comprises at least two thiol groups.
10. The method of claim 1 where the reducing agent has a reduction potential stronger than −0.2V at pH 7 and 25° C.
11. The method of claim 1 where the reducing agent is dithiothreitol or dithioerythritol.
12. The method of claim 11 where the reducing agent is present in the culture medium at a concentration of 2 mM to 18 mM.
13. The method of claim 1 where the heterologous polypeptide or protein is recovered from the cytoplasm of the host cells.
14. 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.
15. The method of claim 1 where step (i) is conducted at a temperature below 37° C.
16. The method of claim 15 where the temperature is between 15° C. and 30° C.
17. 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.
18. The method of claim 1 wherein the host cells further express molecular chaperones.
19. The method of claim 18 where the molecular chaperones are GroEL and GroES.