Recombinant DNA-Methyltransferase M1.BspACI from Bacillus psychrodurans AC: Purification and Properties

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M. V. Tarasova^1,2*, V. V. Kuznetsov³, N. A. Netesova³, D. A. Gonchar¹, and S. Kh. Degtyarev¹

¹SibEnzyme, ul. Akademika Timakova 2/12, 630117 Novosibirsk, Russia; fax: (383) 333-6853; E-mail: tarasovamv@sibenzyme.ru

²Novosibirsk State University, ul. Pirogova 2, 630090 Novosibirsk, Russia

³State Research Center of Virology and Biotechnology “Vector”, 630559 Kol’tsovo, Novosibirsk Region, Russia

^* To whom correspondence should be addressed.

Received May 20, 2010; Revision received June 22, 2010

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A restriction–modification system from Bacillus psychrodurans AC (recognition sequence 5′-CCGC-3′) comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30ºC and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5′-CCGC-3′. The kinetic parameters of M1.BspACI DNA methylation are as follows: K_m for phage λ DNA is 0.053 µM and K_m for S-adenosyl-L-methionine is 5.1 µM. The catalytic constant (k_cat) is 0.095 min^–1.

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KEY WORDS: DNA methyltransferase, Bacillus psychrodurans, enzyme kinetics

DOI: 10.1134/S0006297910120096

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