Cloning, sequencing, overproduction, and purification of M CviBI (GANTC) methyltransferase from Chlorella virus NC-1A

Tin Na J. Kan, Lin Li, Srinivasan Chandrasegaran

Research output: Contribution to journalArticlepeer-review

Abstract

We have cloned and sequenced the cvibIM gene from Chlorella virus NC-1A by selecting for the modification phenotype. The modification gene was cloned on a 7-kb BamHI fragment inserted into the BamHI site of the pUC13 plasmid. The cvibIM gene was localized at the 3′ end of this fragment. Sequencing of this region revealed a large open reading frame that codes for methyltransferase (MTase; symbol M·) (predicting 260 amino acids). M·CviBI (GANTC) aa sequence is homologous to M·Dam(GATC), M·DpnII(GATC), and M·T4 (GATC), and not so to M·HinfI(GANTC), M·HhaII (GANTC), and M·Dpn(GATC). We also describe the use of the polymerase chain reaction technique to alter transcriptional and translational signals surrounding this gene so as to achieve overexpression in Escherichia coli. This construct yields M·CviBI at 2-3% of the total cellular protein. The MTase was purified by phosphocellulose, DEAE, and gel filtration chromatography. Its size by SDS-PAGE is approx. 28 kDa, in good agreement with that predicted from the nucleotide sequence.

Original languageEnglish (US)
Pages (from-to)1-7
Number of pages7
JournalGene
Volume121
Issue number1
DOIs
StatePublished - Nov 2 1992

Keywords

  • Escherichia coli
  • gene cvibIM
  • methylase
  • recombinant DNA
  • restriction endonuclease

ASJC Scopus subject areas

  • Genetics

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