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.

What is claimed is: 
     
         1 .- 18 . (canceled) 
     
     
         19 . A screening method comprising:
 i. identifying which amino acid residues in an amino acid sequence of a restriction endonuclease having star activity are charged amino acids;   ii. mutating one or more codons encoding one or more of the charged residues in a gene sequence encoding the restriction endonuclease;   iii. generating a library of gene sequences having one or more different codon mutations in different charged residues;   iv. obtaining a set of proteins expressed by the mutated gene sequences; and   v. determining a cleavage activity for each protein, and determining a fidelity index for each protein in a predetermined buffer, wherein determining a fidelity index comprises determining the ratio of the highest concentration of protein that does not show star activity to the lowest concentration of protein that completes digestion of the substrate.   
     
     
         20 . A method according to  claim 19 , further comprising:
 i. determining an overall fidelity index improvement factor for proteins belonging to the set of proteins in a defined set of buffers; and/or   ii. mutating codons encoding hydroxylated amino acids, in a same or subsequent step to that of mutating codons for the charged amino acids; and/or   iii. mutating codons encoding amide-containing amino acids, in a same or subsequent step to that of mutating codons for the charged amino acids; and/or   iv. improving the overall fidelity index improvement factor using saturation mutagenesis of one or more of the mutated codons.   
     
     
         21 . A method according to  claim 19 , wherein the codons are mutated to an alanine, except for tyrosine which is mutated to a phenylalanine. 
     
     
         22 . A method according to  claim 20 , wherein the codons are mutated to an alanine, except for tyrosine which is mutated to a phenylalanine.