Characterization of DNA binding activities of over-expressed KpnI restriction endonuclease and modification methylase

The genes encoding the KpnI restriction endonuclease and methyltransferase from Klebsiella pneumoniae have been cloned and expressed in Escherichia co li using a two plasmid strategy. The gene for KpnI methylase with its promo ter was cloned and expressed in pACYC184. Even though the methylase clone i s in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone of KpnI endonuclease, pETRK was enginee red by cloning the R gene into the T7 expression system. This strategy resu lted in over-expression of KpnI endonuclease to about 15-30% of cellular pr otein. Both the enzymes were purified using a single chromatographic step t o apparent homogeneity. The yield of purified endonuclease and methylase fr om one liter of culture was approximately 30 and 6 mg respectively. Electro phoretic mobility shift assays show that both the enzymes are capable of bi nding to specific recognition sequence in the absence of any cofactors. The complexes of KpnI methyl transferase and endonuclease with their cognate s ite exhibit distinctive behaviour with respect to ionic requirement.