Methods for inducible expression of polypeptides
The present invention is an inducible coexpression system, capable of controlled induction of expression of each gene product.
1. A method of producing a polypeptide, the method comprising: growing a culture of a host cell, wherein the host cell comprises at least one extrachromosomal expression construct comprising a first inducible promoter selected from the group consisting of an Escherichia coli sugar-inducible promoter and an Escherichia coli propionate-inducible promoter, and at least one polynucleotide sequence encoding a polypeptide, wherein at least one said polynucleotide sequence is to be transcribed from said first inducible promoter, wherein the polypeptide lacks a signal peptide,
wherein the host cell comprises at least one extrachromosomal expression construct comprising a second inducible promoter, selected from the group consisting of an Escherichia coli sugar-inducible promoter and an Escherichia coli propionate-inducible promoter, that is inducible by at least one inducer that is different than that of said first inducible promoter,
wherein neither of said inducible promoters is a lactose-inducible promoter,
wherein the host cell has an altered gene function of at least one gene that affects the reduction/oxidation environment of the host cell cytoplasm, and
wherein the host cell has a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one said inducible promoter; and
adding an inducer of said first inducible promoter to the culture.
2. The method of claim 1 wherein said expression construct comprising said first inducible promoter, and said expression construct comprising said second inducible promoter, are maintained on the same extrachromosomal polynucleotide.
3. The method of claim 1 wherein at least one inducible promoter is selected from the group consisting of an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, and a xylose-inducible promoter.
4. The method of claim 3 wherein at least one inducible promoter is an L-arabinose-inducible promoter.
5. The method of claim 3 wherein at least one inducible promoter is selected from the group consisting of the araBAD promoter, the prpBCDE promoter, the rhaSR promoter, and the xlyA promoter.
6. The method of claim 1 wherein the polypeptide lacking a signal peptide comprises a polypeptide sequence selected from the group consisting of (a) a polypeptide chain of mature insulin; (b) a botulinum neurotoxin heavy chain; (c) a botulinum neurotoxin light chain; (d) a chaperone; (e) an immunoglobulin heavy chain; (f) an immunoglobulin light chain; (g) a manganese peroxidase; (h) an arabinose-utilization enzyme; (i) a xylose-utilization enzyme; (j) a lignin-degrading peroxidase; and (k) a fragment of any of (e)-(g).
7. The method of claim 1 wherein the at least one gene that affects the reduction/oxidation environment of the host cell cytoplasm is selected from the group consisting of gor, gshA, gshB, and trxB.
8. The method of claim 1 wherein the host cell expresses a mutant form of AhpC.
9. The method of claim 1 wherein at least one gene encoding a protein that metabolizes an inducer of at least one said inducible promoter is selected from the group consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB.
10. The method of claim 1 wherein the host cell comprises at least one expression construct encoding at least one disulfide bond isomerase protein.
11. The method of claim 1 wherein the host cell comprises at least one polynucleotide encoding a form of DsbC lacking a signal peptide.
12. The method of claim 1 wherein the host cell comprises at least one polynucleotide encoding Erv1p.
13. The method of claim 1 wherein the host cell has an alteration of gene function of at least one gene encoding a transporter protein for an inducer of at least one said inducible promoter.
14. The method of claim 13 wherein at least one gene encoding a transporter protein is selected from the group consisting of araE, araF, araG, araH, rhaT, xylF, xylG, and xylH.
15. The method of claim 1 wherein the host cell is a prokaryotic cell.
16. The method of claim 15 wherein the host cell is E. coli.
17. The method of claim 1 wherein at least one inducer is selected from the group consisting of a sugar and propionate.
18. The method of claim 17 wherein at least one inducer is selected from the group consisting of L-arabinose, L-rhamnose, D-xylose, and propionate.
19. The method of claim 18 wherein at least one inducer is L-arabinose.
20. The method of claim 18 wherein at least one inducer is present in the composition at a concentration per host cell density (OD 600 ) of 0.5, and is selected from the group consisting of L-arabinose at a concentration between 2% and 0.001%; L-rhamnose at a concentration between 5% and 0.002%; D-xylose at a concentration between 5% and 0.002%; and propionate at a concentration between 1 M and 1 mM.