High Fidelity Restriction Endonucleases

Abstract

Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.

1 - 43 . (canceled) 
     
     
         44 . A composition comprising a variant KpnI restriction endonuclease having reduced star activity, wherein the variant KpnI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent KpnI restriction endonuclease by:
 (a) an amino acid substitution at one or more positions corresponding to positions 2, 16, 119, 132 and 134 in SEQ ID NO:87; or   (b) amino acid substitutions at positions corresponding to positions 16 and 148 in SEQ ID NO:87.   
     
     
         45 . The composition of  claim 44 , wherein the variant KpnI restriction endonuclease comprises an amino acid substitution at an amino acid position corresponding to position 16 in SEQ ID NO:87. 
     
     
         46 . The composition of  claim 45 , wherein the amino acid substitution is D16N. 
     
     
         47 . The composition of  claim 44 , wherein the variant KpnI restriction endonuclease comprises amino acids substitutions at amino acid positions corresponding to positions 16 and 148 in SEQ ID NO:87. 
     
     
         48 . The composition of  claim 47 , wherein the amino acid substitutions are D16N and D148E. 
     
     
         49 . The composition of  claim 44 , wherein the variant KpnI restriction endonuclease comprises amino acids substitutions at amino acid positions corresponding to positions 16, 132 and 148 in SEQ ID NO:87. 
     
     
         50 . The composition of  claim 49 , wherein the amino acid substitutions are D16N, E132A and D148E.