High fidelity restriction endonucleases
View Patent ↗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. A composition, comprising a variant AgeI restriction endonuclease having reduced star activity, wherein the variant AgeI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent AgeI restriction endonuclease by a mutation at an amino acid position corresponding to position 139 in SEQ ID NO:79.
2. A composition according to claim 1 , wherein the mutation is R139A.
3. A composition comprising a variant AgeI restriction endonuclease having reduced star activity, wherein the variant AgeI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent AgeI restriction endonuclease by a mutation at an amino acid position corresponding to position 201 in SEQ ID NO:79.
4. A composition according to claim 3 , wherein the mutation is S201A.