Protein expression systems
View Patent ↗The present invention provides an improved expression system for the production of recombinant polypeptides utilizing auxotrophic selectable markers. In addition, the present invention provides improved recombinant protein production in host cells through the improved regulation of expression.
1. A process for the production of a recombinant polypeptide, the process comprising:
a) providing an auxotrophic Pseudomonas fluorescens host cell, wherein said host cell comprises a lacI gene inserted into the chromosome;
b) introducing into said host cell a nucleic acid construct comprising:
i) a first nucleotide sequence encoding said recombinant polypeptide, wherein said first nucleotide sequence is operably linked to a promoter capable of directing expression of the first nucleotide sequence in said Pseudomonas fluorescens host cell; and
ii) a second nucleotide sequence encoding an auxotrophic selection marker wherein said second nucleotide sequence is operably linked to a promoter, wherein the auxotrophic selection marker is a polypeptide that restores prototrophy to the auxotrophic host cell; and
c) growing the auxotrophic Pseudomonas fluorescens host cell comprising the first and the second nucleotide sequences on a medium that lacks a metabolite for which the host cell is auxotrophic, wherein the auxotrophic selection marker encoded by the second nucleotide sequence and the recombinant polypeptide encoded by the first nucleotide sequence are expressed, and wherein the auxotrophic Pseudomonas fluorescens host cell is: auxotrophic for uracil, wherein the polypeptide that restores prototrophy is orotidine-5′-phosphate decarboxylase; auxotrophic for proline, wherein the polypeptide that restores prototrophy is Δ 1 -pyrroline-5-carboxylate reductase; or both; and: wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for uracil, the metabolite is uracil; wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for proline, the metabolite is proline; and wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for uracil and proline, the metabolites are uracil and proline;
wherein the auxotrophic Pseudomonas fluorescens host cell expressing the first and the second nucleotide sequences is grown to a density of at least about 20 grams per liter to about 150 grams per liter.
2. The process of claim 1 , wherein the host cell is auxotrophic for the uracil and the proline.
3. The process of claim 1 , wherein when the enzyme is orotidine-5′-phosphate decarboxylase, the polypeptide is encoded by the nucleic acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3.
4. The process of claim 1 , wherein when the polypeptide is Δ 1 -pyrroline-5-carboxylate reductase, the enzyme is encoded by the nucleic acid sequence as set forth in SEQ ID NO:6 or SEQ ID NO:8.
5. The process of claim 1 , wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for uracil the cell comprises a pyrF gene knockout.
6. The process of claim 5 , wherein the pyrF gene comprises the nucleic acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3.
7. The process of claim 1 , wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for proline the cell comprises a proC gene knockout.
8. The process of claim 7 , wherein the proC gene comprises the nucleic acid sequence as set forth in SEQ ID NO:6 or SEQ ID NO:8.
9. The process of claim 1 , wherein when the auxotrophic P. fluorescens host cell is auxotrophic for uracil and proline the cell comprises a pyrF and a proC gene knockout.
10. The process of claim 9 , wherein the pyrF gene comprises the nucleic acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3, and wherein the proC gene comprises the nucleic acid sequence as set forth in SEQ ID NO:6 or SEQ ID NO:8.
11. The process of claim 1 , wherein said host cell lacks a nucleic acid sequence encoding an antibiotic selection marker and wherein said host cell is grown in the absence of an antibiotic.
12. The process of claim 1 , wherein the lacI gene insert is other than as part of a PlacI-lacI-lacZYA operon.
13. The process of claim 1 , wherein the lacI gene is selected from the group consisting of lacI, lacIQ, and lacIQ1 .
14. The process of claim 1 , wherein the nucleic acid construct further comprises at least one lac operator sequence.
15. The process of claim 14 , wherein the at least one lac operator sequence comprises a first lac operator sequence located 5′ of said promoter, and a second lac operator sequence located 3′ of said promoter.
16. The process of claim 14 , wherein the at least one lac operator sequence is a lacOid sequence.
17. The process of claim 14 , wherein the lacOid sequence is selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:59.
18. The process of claim 1 , wherein the lacI gene is inserted in a levansucrase locus.
19. The process of claim 1 , wherein said cell density is at least about: 25 grams per liter; 30 grams per liter; 35 grams per liter; 40 grams per liter; 45 grams per liter; 50 grams per liter; 60 grams per liter; 70 grams per liter; 80 grams per liter; 90 grams per liter; 100 grams per liter; 110 grams per liter; 120 grams per liter; 130 grams per liter; 140 grams per liter; or is about 150 grams per liter.
20. The process of claim 1 , wherein said cell density is about 20 grams per liter to 70 grams per liter measured as dry cell weight.