Polypeptide composition and an immunological analysis of DNA methyltransferases from different species

The MspI restriction-modification system, which recognizes the sequence 5'-CCGG-3', has been previously cloned and sequenced (1). We subcloned the methyltransferase gene (M.MspI) downstream of the ptac promoter in the multicopy vector pUC119 and overexpressed it in E. coli. Upon induction with IPTG, M.MspI constitutes more than 10% of cellular protein. A scheme has been devised to purify large amounts of biologically active M.MspI to apparent homogeneity from these overexpressing E. coli cells. Approximately 0.8 mg of pure M.MspI per gram of cells (wet weight) can be obtained. The apparent molecular weight of M.MspI 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.MspI cross-react with the DNA-methyltransferases of several other restriction-modification systems.

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

Dubey

Mollet

Roberts

1992

Nucl Acids Res

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MINIREVIEW: A Role for DNA Methylation in Vertebrate Gene Expression?

Strobl

1990

Molecular Endocrinology

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Polypeptide composition and an immunological analysis of DNA methyltransferases from different species

Cited by 2 publications

References 19 publications

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

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

MINIREVIEW: A Role for DNA Methylation in Vertebrate Gene Expression?

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