Variant of KpnI restriction endonuclease
View Patent ↗Methods are provided for making restriction endonucleases with reduced star activity by one or more targeted mutations to a catalytic site within the restriction endonuclease. Examples of modifications to restriction endonucleases with significant sequence identity with KpnI are provided and reduced star activity demonstrated.
1. A variant KpnI restriction endonuclease having at least 90% sequence identity with SEQ ID NO: 13 wherein
the amino acid at the position corresponding to amino acid 163 of SEQ ID NO: 13 is not Asp (D);
the amino acid at the position corresponding to amino acid 165 of SEQ ID NO: 13 is not Lys (K);
the amino acid at the position corresponding to amino acid 148 of SEQ ID NO: 13 is not a Asp (D);
the amino acid at the position corresponding to amino acid 175 of SEQ ID NO: 13 is not Gln (Q);
or
the amino acids at the positions corresponding to amino acids 163 and 165 of SEQ ID NO: 13 are not Asp (D) and Lys (K), respectively.
2. The variant KpnI restriction endonuclease according to claim 1 having an amino acid sequence consisting essentially of SEQ ID NO: 13, wherein
the amino acid at the position corresponding to amino acid 163 of SEQ ID NO: 13 is not Asp (D);
the amino acid at the position corresponding to amino acid 165 of SEQ ID NO: 13 is not Lys (K);
the amino acid at the position corresponding to amino acid 148 of SEQ ID NO: 13 is not a Asp (D);
the amino acid at the position corresponding to amino acid 175 of SEQ ID NO: 13 is not Gln (Q);
or
the amino acids at the positions corresponding to amino acids 163 and 165 of SEQ ID NO: 13 are not Asp (D) and Lys (K), respectively.