Purification of the KpnI DNA methyltransferase and photolabeling of the enzyme with S-adenosyl-l-methionine

Csaba Finta, Urszula Sulima, Pál Venetianer, Antal Kiss

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

An Escherichia coli strain overproducing the KpnI DNA methyltransferase (M·KpnI) was constructed by cloning the kpnIM gene downstream from the inducible T7 phage Φ promoter. A method involving three chromatographic steps has been developed to purify M·KpnI to homogeneity. The purified enzyme has a pH optimum around 7.3 and is inhibited by salts. M·KpnI can be photolabeled by UV-irradiation of the enzyme in the presence of S-adenosyl-l-[methyl-3h]methionine ([methyl-3h]AdoMet). Photolabeling results from a specific interaction between M·KpnI and AdoMet, as indicated by the dependence of photolabeling on native enzyme conformation and by the inhibitory effect of the AdoMet analogs, sinefungin and S-adenosyl-l-homocysteine (AdoHcy).

Original languageEnglish
Pages (from-to)65-69
Number of pages5
JournalGene
Volume164
Issue number1
DOIs
Publication statusPublished - Oct 16 1995

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Keywords

  • DNA methylation
  • UV crosslinking
  • fluorography
  • overexpression
  • restriction-modification
  • sinefungin

ASJC Scopus subject areas

  • Genetics

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