101. [Recombinant DNA-methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: purification, propeties and amino acids sequence analysis].
- Author
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Tomilova IuÉ, Kuznetsov VV, Abdurashitov MA, Netesova NA, and Degtiarev SKh
- Subjects
- Bacterial Proteins metabolism, Bacteriophage lambda metabolism, DNA Modification Methylases metabolism, DNA, Viral metabolism, Escherichia coli genetics, Geobacillus stearothermophilus genetics, Hot Temperature, Kinetics, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Bacterial Proteins chemistry, Bacteriophage lambda chemistry, DNA Methylation, DNA Modification Methylases chemistry, DNA, Viral chemistry, Geobacillus stearothermophilus enzymology
- Abstract
The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5'-GCATC-3') in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW, that carries a tandem of thermo inducible promoters P(R)/P(L) from phage lambda. Highly purified enzyme has been isolated by chromatography on various resins from E. coli cells where it accumulates in a soluble form. Study of M1.Bst19I properties has revealed that enzyme has a temperature optimum 50 degrees C and demonstrates the maximum activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5'-GCATC-3'. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for lambda DNA is 0.68 +/- 0.07 microM, Km for S-adenosil-L-methionine is 2.02 +/- 0.31 microM. Catalytical constant (kcat) is 1.8 +/- 0.05 min(-1). Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and others alpha-N6-DNA methyltransferases has allowed to suppose a presence of two types of the enzymes containing a triplet ATG or ATC in the recognition sequence.
- Published
- 2010