Purification and characterization of the MspI DNA methyltransferase cloned and overexpressed in E. coli

Dubey, A. K., Mollet, B., Roberts, R. J. (1992) Purification and characterization of the MspI DNA methyltransferase cloned and overexpressed in E. coli. Nucleic Acids Res, 20 (7). pp. 1579-85. ISSN 0305-1048 (Print)0305-1048 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1579450
DOI: 10.1093/nar/20.7.1579

Abstract

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.

Item Type: Paper
Uncontrolled Keywords: Blotting, Western Cloning, Molecular DNA-Cytosine Methylases/*genetics/isolation & purification/metabolism Escherichia coli/*enzymology/genetics Gene Expression Regulation, Bacterial/drug effects Isopropyl Thiogalactoside/pharmacology Plasmids/genetics
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA methylation
organism description > bacteria > escherichia coli
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > methyltransferase
CSHL Authors:
Communities: CSHL labs > Roberts lab
Depositing User: Matt Covey
Date Deposited: 23 Sep 2015 15:55
Last Modified: 23 Sep 2015 15:55
PMCID: PMC312241
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31841

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