Process for Producing Polypeptides

Abstract

A process is described for producing a polypeptide heterologous to E. coli wherein E. coli cells comprising nucleic acid encoding the polypeptide are cultured in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed. The polypeptide is then recovered from the cells.

1 . A process for producing a polypeptide heterologous to  E. coli  comprising (a) culturing  E. coli  cells comprising nucleic acid encoding the polypeptide in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed, and (b) recovering the polypeptide from the cells.  
     
     
         2 . The process of  claim 1  wherein the organophosphate is a glycerophosphate.  
     
     
         3 . The process of  claim 2  wherein the glycerophosphate is an alpha-glycerophosphate or beta-glycerophosphate, or a mixture thereof.  
     
     
         4 . The process of  claim 3  wherein the glycerophosphate is a mixture of glycerol-2-phosphate and glycerol-3-phosphate or is glycerol-3-phosphate.  
     
     
         5 . The process of  claim 1  wherein the culturing takes place in a shake flask or fermentor.  
     
     
         6 . The process of  claim 1  wherein the polypeptide is recovered from the cytoplasm, periplasm or culture medium of the cells.  
     
     
         7 . The process of  claim 1  wherein expression of the nucleic acid is regulated by an inducible promoter.  
     
     
         8 . The process of  claim 7  wherein the inducible promoter is the alkaline phosphatase promoter.  
     
     
         9 . The process of  claim 7  wherein the inducible promoter is the tac promoter.  
     
     
         10 . The process of  claim 7  wherein the inducible promoter is the T7 promoter.  
     
     
         11 . The process of  claim 7  wherein expression of the nucleic acid begins while in the active growth phase of the culturing step.  
     
     
         12 . The process of  claim 1  wherein the  E. coli  is deficient in chromosomal phoA.  
     
     
         13 . The process of  claim 1  wherein the  E. coli  is wild type with respect to chromosomal gipT.  
     
     
         14 . The process of  claim 1  wherein the  E. coli  is deficient in chromosomal glpT.  
     
     
         15 . The process of  claim 1  wherein the  E. coli  is deficient in chromosomal phoA and glpT.  
     
     
         16 . The process of  claim 15  wherein the  E. coli  is not deficient in chromosomal ugp.  
     
     
         17 . The process of  claim 1  wherein the polypeptide is a eukaryotic polypeptide.  
     
     
         18 . The process of  claim 1  wherein the polypeptide is a mammalian polypeptide.  
     
     
         19 . The process of  claim 1  wherein the polypeptide is insulin-like growth factor-1.  
     
     
         20 . The process of  claim 19  wherein the feed rate of the organophosphate is from about 1 to 7 mmoles/hour per about 8-10 liters and the culturing takes place in a 10-liter fermentor.  
     
     
         21 . The process of  claim 20  wherein the feed rate of the organophosphate is from about 2 to 6 mmoles/hour per about 8-10 liters.  
     
     
         22 . The process of  claim 21  wherein the feed rate of the organophosphate is from about 3 to 4 mmoles/hour per about 8- 10 liters.  
     
     
         23 . The process of  claim 1  wherein the polypeptide is Apo2L.  
     
     
         24 . The process of  claim 23  wherein the feed rate of the organophosphate is from about 4 to 17 mmoles/hour per about 8-10 liters and the culturing takes place in a 10-liter fermentor.  
     
     
         25 . The process of  claim 24  wherein the feed rate is from about 6 to 16 mmole/hour per about 8-10 liters.  
     
     
         26 . The process of  claim 25  wherein the feed rate is from about 8 to 15 mmole/hour per about 8-10 liters.  
     
     
         27 . The process of  claim 26  wherein the feed rate is from about 10 to 14 mmole/hour per about 8-10 liters.  
     
     
         28 . The process of  claim 1  wherein an inorganic phosphate is also present during the culturing step.  
     
     
         29 . The process of  claim 28  wherein the ratio of inorganic phosphate to organophosphate ranges from about 1:10 to 1:0.25.  
     
     
         30 . The process of  claim 29  wherein the polypeptide is Apo2L and the ratio is about 1:3 to 1:0.5.  
     
     
         31 . The process of  claim 30  wherein the ratio is about 1:1.