Process for producing igf-1 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.

What is claimed is: 
     
         1 . A process for producing IgF-1 polypeptide recombinantly comprising: (a) culturing  E. coli  cells comprising a heterologous nucleic acid encoding the IgF-1 in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed, and (b) recovering the IgF-1 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 glpT. 
     
     
         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 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. 
     
     
         18 . The process of  claim 17  wherein the feed rate is from about 6 to 16 mmole/hour per about 8-10 liters. 
     
     
         19 . The process of  claim 18  wherein the feed rate is from about 8 to 15 mmole/hour per about 8-10 liters. 
     
     
         20 . The process of  claim 19  wherein the feed rate is from about 10 to 14 mmole/hour per about 8-10 liters. 
     
     
         21 . The process of  claim 1 , wherein an inorganic phosphate is also present in the culture medium during the culturing step. 
     
     
         22 . The process of  claim 21  wherein the ratio of inorganic phosphate to organophosphate ranges from about 1:10 to 1:0.25 during the culturing step. 
     
     
         23 . The process of  claim 22  wherein the ratio is about 1:3 to 1:0.5. 
     
     
         24 . The process of  claim 23  wherein the ratio is about 1:1.