Purification and characterization of theMspl DNA methyltransferase cloned and overexpressed inE.coli

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Abstract
The Mspl restriction-modification system, which recognizes the sequence 5'-CCGG-3', has been previously cloned and sequenced (1). We subcloned the methyltransferase gene (M.Mspl) downstream of the ptac promoter In the multicopy vector pUC119 and overexpressed it in E. coli. Upon induction with IPTG, M.Mspl constitutes more than 10% of cellular protein. A scheme has been devised to purify large amounts of biologically active M.Mspl to apparent homogeneity from these overexpressing E. coli cells. Approximately 0.8 mg of pure M.Mspl per gram of cells (wet weight) can be obtained. The apparent molecular weight of M.Mspl is 49 kD, by SDS gel electrophoresis and 48–54 kD by gel filtration. At low concentrations (less than 0.4 mg/ml), the methyltransferase is a monomer in solution but at higher concentrations (greater than 3.0 mg/ml) it exists predominantly as a dimer. Polyclonal antibodies raised against M.Mspl cross-react with the DNA-methyltransferases of several other restriction-modification systems.