Methods of increasing protein expression levels

Abstract

The present invention relates to methods of increasing protein expression levels whereby at least one amino acid in a protein amino acid sequence is substituted for the amino acid, proline. Preferably, the substitution occurs within 15 amino acids, more preferably within 10 amino acids and most preferably within 5 amino acids of a cysteine amino acid residue. The present invention not only includes methods for polypeptides with proline substitutions, but also polynucleotides with codon substitutions for which a codon for any amino acid, except proline, is substituted for a codon encoding for proline.

1 . An isolated nucleic acid comprising a sequence that encodes a mutant of a native polypeptide wherein the native polypeptide consists of the amino acid sequence of SEQ ID NO:1, and wherein the amino acid at position 23 or the amino acid at position 76 of SEQ ID NO:1 is substituted with proline and wherein said mutant exhibits increased expression compared with that of the native polypeptide.  
     
     
         2 . The nucleic acid of  claim 1 , wherein the amino acid at position 23 of SEQ ID NO:1 is substituted with proline.  
     
     
         3 . The nucleic acid of  claim 1 , wherein the amino acid at position 76 of SEQ ID NO:1 is substituted with proline.  
     
     
         4 . The nucleic acid of  claim 1 , wherein the native polypeptide is encoded by the sequence of SEQ ID NO:2.  
     
     
         5 . The nucleic acid of  claim 1 , further comprising a sequence that encodes a signal peptide.  
     
     
         6 . An expression vector comprising the nucleic acid of any one of claims  1 - 5 , wherein said nucleic acid is operatively linked to an expression control sequence.  
     
     
         7 . A host cell comprising the expression vector of  claim 6.