Methods for making proteins containing free cysteine residues
The present invention relates to novel methods of making soluble proteins having free cysteines in which a host cell is exposed to a cysteine blocking agent. The soluble proteins produced by the methods can then be modified to increase their effectiveness. Such modifications include attaching a PEG moiety to form pegylated proteins.
1. A method for expressing a recombinant interferon alpha protein in a Escherichia coli ( E. coli ) host cell comprising the steps of:
(a) obtaining an E. coli strain containing a DNA sequence encoding the N-terminus of the mature form of the interferon alpha protein joined to the C-terminus of the E. coli STII signal sequence;
(b) culturing the E. coli strain in a culture media wherein the pH is lower than pH 6.5; and
(c) inducing expression of the interferon alpha protein; wherein the interferon alpha protein is secreted into the E. coli periplasm.
2. The method of claim 1 , further comprising the steps of (d) lysing the induced E. coli cells and (e) isolating the interferon alpha protein from the E. coli host cell proteins.
3. The method of claim 2 , wherein the interferon alpha protein is isolated from E. coli host cell proteins following osmotic shock of the E. coli cells.
4. The method of claim 3 , wherein the interferon alpha protein is isolated from the supernatant fraction following centrifugation of the osmotic shock lysate from the osmotic shock treated E. coli cells.
5. The method of claim 4 , wherein the interferon alpha protein in the supernatant fraction is isolated by column chromatography.
6. The method of claim 1 , wherein the culture media pH is pH 6.0 or lower.
7. The method of claim 1 , wherein the culture media pH is between pH 5.5 and pH 6.0.
8. The method of claim 1 , wherein the culture media pH is pH 5.5.
9. The method of claim 1 , wherein step (b) of culturing occurs in the presence of 2-(N-morpholino)ethanesulfonic acid (MES).
10. The method of claim 1 , wherein the STII signal sequence is cleaved from the mature interferon alpha protein.
11. The method of claim 1 , wherein the interferon alpha protein is human interferon alpha.
12. The method of claim 1 , wherein the interferon alpha protein is interferon alpha-2.
13. The method of claim 1 , wherein the interferon alpha protein is a cysteine mutein of interferon alpha.
14. The method of claim 13 , wherein the interferon alpha protein is human interferon alpha-2 and wherein said interferon alpha-2 protein contains a cysteine residue substituted for an amino acid selected from the group consisting of D2, L3, Q5, S8, H7, N45, Q101, G104, T106, V105, E107, M111, S163, K164, and E165.
15. The method of claim 13 , wherein the interferon alpha protein is human interferon alpha-2 and wherein the interferon alpha-2 protein contains a cysteine residue substituted for Q5.
16. The method of claim 13 , wherein the interferon alpha protein is human interferon alpha-2 and wherein the interferon alpha-2 protein contains a cysteine residue substituted for M111.
17. The method of claim 13 , wherein the interferon alpha protein is modified with polyethylene glycol at a free cysteine.
18. The method of claim 17 , wherein the polyethylene glycol is a cysteine-reactive polyethylene glycol.
19. The method of claim 17 , wherein the polyethylene glycol is a maleimide polyethylene glycol.