Expression of soluble factor viii proteins in bacteria

Abstract

The present invention provides enhanced methods of producing soluble Factor VIII proteins in microorganisms that have an oxidizing environment.

1 . A method of producing a soluble Factor VIII protein in a microorganism, wherein the microorganism has an oxidizing environment,
 the method comprising the steps of   a) expressing in the microorganism a nucleic acid that encodes the Factor VIII protein; and   b) growing the microorganism under conditions that allow production of the soluble Factor VIII protein.   
     
     
         2 . The method of  claim 1 , wherein the microorganism is an  E. coli.    
     
     
         3 . The method of  claim 2 , wherein the  E. coli  has a mutation in a trxB gene and a gor gene. 
     
     
         4 . The method of  claim 2 , wherein the nucleic acid is expressed under the control of an inducible promoter. 
     
     
         5 . The method of  claim 4 , wherein expression of the nucleic acid is induced at a temperature lower than an optimal growth temperature. 
     
     
         6 . The method of  claim 1 , wherein the microorganism has a mutation in an endogenous reductase nucleic acid. 
     
     
         7 . The method of  claim 1 , wherein the microorganism is grown at a temperature lower than an optimal growth temperature. 
     
     
         8 . The method of  claim 1 , further comprising the step of isolating the soluble factor VIII protein. 
     
     
         9 . The method of  claim 1 , wherein the soluble Factor VIII protein is produced on a commercial scale. 
     
     
         10 . The method of  claim 1 , wherein the Factor VIII protein comprises a purification tag. 
     
     
         11 . The method of  claim 10 , wherein the purification tag is removed from the Factor VIII protein. 
     
     
         12 . The method of  claim 1 , wherein the microorganism comprises a heterologous protein disulfide isomerase (PDI). 
     
     
         13 . The method of  claim 1 , wherein the microorganism comprises a heterologous chaperone protein. 
     
     
         14 . The method of  claim 1 , wherein the Factor VIII protein exhibits enzymatic or biological activity. 
     
     
         15 . The method of  claim 1 , wherein all or a portion of a B-domain is deleted from the Factor VIII protein.