Method of making proteins in transformed yeast cells

Abstract

The present invention relates to a method for expressing heterologous proteins or polypeptides in yeast by culturing a transformed yeast strain which does not contain a functional antibiotic resistance marker gene.

1 . Recombinant yeast expression vector being unable to confer antibiotic resistance to bacteria cells said vector comprising a gene coding for a heterologous gene and a antibiotic resistance marker gene which has been made non functional by in vitro modification.  
     
     
         2 . Recombinant yeast expression vector according to  claim 1  wherein the antibiotic marker gene is the AMP gene.  
     
     
         3 . Method for making a desired polypeptide or protein comprising culturing a yeast strain comprising a vector according to claims  1 - 2  under suitable conditions and isolating the desired product from the culture medium.  
     
     
         4 . Method for making a desired polypeptide or protein comprising culturing under suitable conditions a yeast strain containing a yeast expression plasmid in which plasmid a functional antibiotic marker gene used for initial cloning steps in bacteria has been made non-functional by in vitro deletion of part of the marker gene or the whole marker gene before insertion into the yeast host.  
     
     
         5 . Method according to  claim 4 , comprising inserting suitable restriction sites around the antibiotic resistance marker gene and deleting the marker gene by in vitro treatment with suitable restriction enzymes.  
     
     
         6 . Method according to claims  3 - 5 , wherein the yeast strain is a Saccharomyces strain, preferably a  Saccharomyces cerevisiae  strain.  
     
     
         7 . Transformed yeast cell comprising a vector according to claims  1 - 2 .  
     
     
         8 . Transformed yeast cell according to  claim 7  being a Saccharomyces strain, preferable a  Saccharomyces cerevisiae  strain.