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

摘要

The genes encoding the KpnI restriction endonuclease and methyltransferase from Klebsiella pneumoniae have been cloned and expressed in Escherchia coli using a two plasmid strategy. The gene for KpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone of KpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression of KpnI endonuclease to about 15-30% of cellular protein. Both the enzymes were purified using a single chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes of KpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.