Method for Reducing Star Activity in Restriction Endonucleases

Abstract

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.

What is claimed is: 
     
         1 . A KpnI restriction endonuclease in an effective cleavage buffer, the KpnI having reduced star activity for substrate DNA, compared with star activity of KpnI for the same substrate in 10 mM Bis Tris Propane-HCl, 10mM MgCl 2 , 1 mM dithiothreitol pH 7.0 at 25° C., as determined by gel electrophoresis. 
     
     
         2 . A KpnI restriction endonuclease according to  claim 1 , wherein the effective cleavage buffer contains calcium ions. 
     
     
         3 . (withdrawn and currently amended) A modified KpnI restriction endonuclease having a catalytic motif (SEQ ID NO:16), wherein one or more amino acids in the motif are mutated, the modified KpnI restriction endonuclease having reduced star activity in a buffer for substrate DNA, compared with star activity of unmodified KpnI in the same buffer as determined by gel electrophoresis. 
     
     
         4 . A KpnI restriction endonuclease according to  claim 3 , wherein the one or more amino acid mutations is a single mutation selected from D163 and K165. 
     
     
         5 . A KpnI restriction endonuclease according to  claim 4 , wherein the one or more amino acid mutations is a single mutation selected from D163I and K165A. 
     
     
         6 . A KpnI restriction endonuclease according to  claim 3 , wherein the one or more amino acid mutations are two amino acid mutations. 
     
     
         7 . A KpnI restriction endonuclease according to  claim 6 , wherein the two amino acid mutations are D163 and K165. 
     
     
         8 . A KpnI restriction endonuclease according to  claim 7 , wherein the two amino acid mutations are D163I and K165A. 
     
     
         9 . A DNA encoding the restriction endonuclease of  claim 3 ,  5  or  7 . 
     
     
         10 . A host cell transformed with DNA of  claim 9 . 
     
     
         11 . A method for reducing star activity of a restriction endonuclease, comprising:
 (a) creating one or more mutations targeted to DNA encoding a catalytic site within a DNA encoding the restriction endonuclease;   (b) transforming a host cell with the DNA encoding the mutated restriction endonuclease;   (c) assaying the mutated restriction endonuclease produced by the transformed host cells for reduced star activity; and   (d) selecting the restriction endonuclease with reduced star activity.   
     
     
         12 . A method according to  claim 11 , wherein the restriction endonuclease has at least 50% sequence homology with KpnI. 
     
     
         13 . A method according to  claim 11 , wherein the restriction endonuclease has at least 60% sequence homology with KpnI. 
     
     
         14 . A method according to  claim 11 , wherein the restriction endonuclease has at least 70% sequence homology with KpnI. 
     
     
         15 . A method according to  claim 11 , wherein the restriction endonuclease has at least 80% sequence homology with KpnI. 
     
     
         16 . A method according to  claim 11 , wherein the restriction endonuclease has at least 90% sequence homology with KpnI. 
     
     
         17 . A method according to any of  claims 11  through 16 wherein the one or more mutations are targeted to the (SEQ ID NO:16) motif. 
     
     
         18 . A method according to  claims 11  through  17  further comprising two mutations.