Expression of soluble, active eukaryotic glycosyltransferases in prokaryotic organisms

Abstract

The present invention provides enhanced methods of producing soluble, active eukaryotic glycosyltransferases in prokaryotic microorganisms that have an oxidizing environment.

1 . A method of producing a soluble active eukaryotic glycosyltransferase in a prokaryotic microorganism, wherein the prokaryotic microorganism has an oxidizing environment, the method comprising the steps of a) expressing a nucleic acid that encodes the eukaryotic glycosyltransferase in the prokaryotic microorganism; and b) growing the microorganism under conditions that allow expression of the soluble active eukaryotic glycosyltransferase within a cellular compartment of prokaryotic microorganism. 
     
     
         2 . The method of  claim 1 , wherein the eukaryotic glycosyltransferase is a member selected from the group consisting of a eukaryotic N-acetylglucosaminyltransferase I (GnT or GNT), a eukaryotic N-acetylgalactosaminyltransferase (GalNAcT), a eukaryotic galactosyltransferase (GalT), and a eukaryotic sialyltransferase. 
     
     
         3 . The method of  claim 2 , wherein the eukaryotic N-acetylglucosaminyltransferase is; the eukaryotic N-acetylgalactosaminyltransferase is; the eukaryotic galactosyltransferase (GalT) is a eukaryotic β-1,4-galactosyltransferase (GalT1) or a eukaryotic core I galactosyltransferase (Core 1 GalT1); the eukaryotic sialyltransferase is a eukaryotic α(2,3)sialyltransferase (ST3Gal3), a eukaryotic α-N-acetylgalactosaminide α-2,6-sialyltransferase I (ST6GalNAcT1), or a eukaryotic gal β1,3GalNAc α2,3-sialyltransferase (ST3GalI). 
     
     
         4 . The method of  claim 1 , wherein the prokaryotic microorganism is an  E. coli  or a  Pseudomonas  bacterium. 
     
     
         5 . The method of  claim 4 , wherein the  E. coli  has a mutation in a txrB gene and a gor gene and is grown at a temperature between 12-30° C. 
     
     
         6 . The method of  claim 1 , wherein the prokaryotic microorganism has a mutation in an endogenous reductase nucleic acid. 
     
     
         7 . The method of  claim 1 , wherein the prokaryotic 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, active eukaryotic glycosyltransferase. 
     
     
         9 . The method of  claim 1 , wherein the soluble active eukaryotic glycosyltransferase is produced on a commercial scale. 
     
     
         10 . The method of  claim 1 , wherein the soluble active eukaryotic glycosyltransferase comprises a purification tag. 
     
     
         11 . The method of  claim 1 , wherein the prokaryotic microorganism comprises a heterologous protein disulfide isomerase (PDI). 
     
     
         12 . The method of  claim 1 , wherein the prokaryotic microorganism comprises a heterologous chaperone protein. 
     
     
         13 . A soluble active eukaryotic glycosyltransferase made by the method of  claim 1 .