Cloning, purification and characterization of the BseCI DNA methyltransferase from Bacillus stearothermophilus.
The gene (bseCIM) encoding the BseCI DNA methyltransferase (MTase; M.BseCI) from a Bacillus stearothermophilus species was cloned and expressed in Escherichia coli using plasmid vector pBR322. Selection of transformants carrying bseCIM was based on the resistance of the modified plasmid to cleavage by BseCI. The MTase was purified to homogeneity and further characterized. Its size as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and size exclusion chromatography was 68 kDa, suggesting that the MTase exists as a monomer. When phage lambda DNA was used as a substrate, the optimum temperature for MTase activity was determined to be 50-55 degrees C and optimum pH approx. 7.4. M.BseCI is inhibited by concentrations of NaCl and KCl greater than 50 mM, and it does not require Mg2+ for activity. Finally, M.BseCI methylates the 3' adenine residue in the sequence, 5'-ATCGAT-3', similarly to its isoschizomer M.ClaI.
Rina
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Institute of Molecular Biology and Biotechnology, Enzyme Technology Division, Crete, Greece.
Bouriotis
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V
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
C061926:DNA modification methylase Cla I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)