Purification and properties of recombinant DNA methyltransferase M2.BstSE of the BstSEI nickase-modification system

The operon for the Bacillus stearothermophilus SE-589 nickase-modification system (NM.BstSEI, recognition site 5'-GAGTC-3') includes two DNA methyltransferase (M.) genes, bstSEIM1 and bstSEIM2. The gene encoding M2.BstSEI was cloned in pJW and expressed in Escherichia coli cells. M2.BstSEI...

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Bibliographic Details
Published in:Molecular biology (New York) 2009-02, Vol.43 (1), p.8-15
Main Authors: Chernukhin, V. A, Seggewiss, J, Kashirina, Yu. G, Gonchar, D. A, Degtyarev, S. Kh
Format: Article
Language:English
Subjects:
Bacillus stearothermophilus
Biochemistry
Biomedical and Life Sciences
Deoxyribonucleic acid
DNA
E coli
Enzymes
Escherichia coli
Genes
Genomics. Transcriptomics. Proteomics
Human Genetics
Kinetics
Life Sciences
Molecular biology
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Summary:The operon for the Bacillus stearothermophilus SE-589 nickase-modification system (NM.BstSEI, recognition site 5'-GAGTC-3') includes two DNA methyltransferase (M.) genes, bstSEIM1 and bstSEIM2. The gene encoding M2.BstSEI was cloned in pJW and expressed in Escherichia coli cells. M2.BstSEI was purified by chromatography and displayed maximal activity at 55° C and pH 7.5. The enzyme modified adenine in the nickase recognition site 5'-GAGTC-3' and was specific for 5'-GASTC-3' substrates. The kinetic parameters of the methylation reaction were determined. The catalytic constant was 2.2 min⁻¹, and the Michaelis constant was 9.8 nM on T7 DNA and 5.8 μM on SAM.
ISSN:0026-8933
1608-3245
DOI:10.1134/S0026893309010026