Processing of Peptides and Proteins

Abstract

The invention provides novel methionine aminopeptidase enzymes, their use, and related compounds and methods.

1 . An  E. coli  aminopeptidase variant comprising one or more amino acid sequence variations in the substrate defining pockets.  
     
     
         2 . The aminopeptidase variant of  claim 1 , wherein the variant has extended substrate specificity the P1′ position relative to the wild-type  E. coli  aminopeptidase.  
     
     
         2 . The aminopeptidase variant of  claim 2 , wherein the aminopeptidase variant has extended substrate specificity in P1′ position to include Asn, Leu, Ile, Phe, His, Gln or Trp as well as the amino acids allowed in position P1′ by the wild type.  
     
     
         3 . The aminopeptidase variant of  claim 3 , wherein the residues in positions 168, 206 and/or 233 (of the wild type aminopeptidase) are substituted.  
     
     
         5 . The aminopeptidase variant of  claim 4 , wherein the residue in position 168 is substituted.  
     
     
         6 . The aminopeptidase variant of  claim 4 , wherein the residue in position 206 is substituted.  
     
     
         7 . The aminopeptidase variant of  claim 4 , wherein the residue in position 233 is substituted.  
     
     
         8 . The aminopeptidase variant of  claim 4 , wherein the residues in positions 206 and 233 are substituted.  
     
     
         9 . The aminopeptidase variant of  claim 4 , wherein the residues in positions 168 and 206 are substituted.  
     
     
         10 . The aminopeptidase variant of  claim 4 , wherein the residues in positions 168 and 233 are substituted.  
     
     
         11 . The aminopeptidase variant of  claim 4 , wherein the residues in positions 168, 206, and 233 are substituted.  
     
     
         12 . The aminopeptidase variant of  claim 4 , wherein the variant comprises one or more substitutions with amino acid residues selected from the group consisting of Gly, Ala, Ser, Thr, Asn, and Asp.  
     
     
         4 . The aminopeptidase variant of  claim 12 , wherein the substitution is selected from Gly and Ala.  
     
     
         5 . The aminopeptidase variant of claim  13 , wherein the substitution is with an Ala residue.  
     
     
         6 . The aminopeptidase variant of claim  14 , wherein the variant comprises a substitution at position 168 with an Ala residue.  
     
     
         7 . The aminopeptidase variant of claim  14 , wherein the variant comprises a substitution at position 206 with an Ala residue.  
     
     
         8 . The aminopeptidase variant of claim  14 , wherein the variant comprises a substitution at position 233 with an Ala residue.  
     
     
         18 . The aminopeptidase variant of claim  16 , wherein the variant comprises substitutions at positions 206 and 233 with Ala.  
     
     
         19 . The aminopeptidase variant of  claim 1 , wherein the variant comprises SEQ ID NO:3.  
     
     
         20 . The aminopeptidase variant of  claim 1 , wherein the variant comprises SEQ ID NO:5.  
     
     
         21 . The aminopeptidase variant of  claim 1 , wherein the variant comprises SEQ ID NO:7.  
     
     
         22 . The aminopeptidase variant of  claim 1 , wherein the variant comprises SEQ ID NO:9.  
     
     
         23 . A method of removing an initiator methionine from a peptide comprising contacting the peptide with an amino peptidase variant according to  claim 1  under conditions suitable for the aminopeptidase variant to remove the initiator methionine.  
     
     
         24 . The method of  claim 23 , wherein the peptide is selected from (a) a recombinant interleukin-21 (IL-21); (b) an IL-21 variant; or (c) a derivative of either (a) or (b).  
     
     
         25 . The method of  claim 23 , wherein the peptide is recombinant interleukin-20 (IL-20).  
     
     
         26 . The method of  claim 23 , wherein the peptide is recombinant human growth hormone (hGH).  
     
     
         27 . The method of  claim 24 , wherein the peptide is recombinant human IL-21.  
     
     
         28 . A method of purifying a peptide product having a Gln residue in the N-terminus from a first peptide comprising the Gln residue and an initiator Met residue, the method comprising applying Qcyclase to the first peptide and purifying the peptide product obtained thereby based on charge differences.  
     
     
         29 . The method of  claim 28 , wherein the peptide product is purified on a cation exchange column.