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 NcoI restriction endonuclease having reduced star activity, wherein the variant NcoI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent NcoI restriction endonuclease by amino acid substitutions at positions corresponding to positions 2 and 31 in SEQ ID NO:88.
2. The composition according to claim 1 , wherein the substitutions are A2T and R31A.
3. A composition comprising a variant NcoI restriction endonuclease having reduced star activity, wherein the variant NcoI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent NcoI restriction endonuclease by a substitution at an amino acid position corresponding to position 56 in SEQ ID NO:88.
4. The composition according to claim 3 , wherein the substitution is D56A.
5. A composition comprising a variant NcoI restriction endonuclease having reduced star activity, wherein the variant NcoI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent NcoI restriction endonuclease by a substitution at an amino acid position corresponding to position 143 in SEQ ID NO:88.
6. The composition according to claim 5 , wherein the substitution is H143A.
7. A composition comprising a variant NcoI restriction endonuclease having reduced star activity, wherein the variant NcoI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent NcoI restriction endonuclease by a substitution at an amino acid position corresponding to position 166 in SEQ ID NO:88.
8. The composition according to claim 7 , wherein the substitution is E166A.
9. A composition comprising a variant NcoI restriction endonuclease having reduced star activity, wherein the variant NcoI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent NcoI restriction endonuclease by a substitution at an amino acid position corresponding to position 212 in SEQ ID NO:88.
10. The composition according to claim 9 , wherein the substitution is R212A.
11. A composition comprising a variant NcoI restriction endonuclease having reduced star activity, wherein the variant NcoI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent NcoI restriction endonuclease by a substitution at an amino acid position corresponding to position 268 in SEQ ID NO:88.
12. The composition according to claim 11 , wherein the substitution is D268A.