METHODS OF INCREASING PROTEIN EXPRESSION LEVELS
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.