Purification and characterization of the MspI DNA methyltransferase cloned and overexpressed in E. coli.
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.
Dubey
A K
AK
Cold Spring Harbor Laboratory, NY 11724.
Mollet
B
B
Roberts
R J
RJ
D015153:Blotting, Western; D003001:Cloning, Molecular; D015257:DNA-Cytosine Methylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics
D007544:Isopropyl Thiogalactoside; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases