Overproduction and purification of the M·HhaII methyltransferase from Haemophilus haemolyticus

Srinivasan Chandrasegaran, Louisa P. Wu, Eduardo Valda, Hamilton O. Smith

Research output: Contribution to journalArticlepeer-review

Abstract

The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-β-d-thiogalactopyranoside results in overproduction of the methyltransferase to about 3% of total cellular protein. The methyltransferase was purified to near electrophoretic homogeneity by phosphocellulose, DEAE, and gel chromatography. Its monomer Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25 kDa, in good agreement with that predicted from the nucleotide sequence. Crystals of the methyltransferase were obtained in the presence of a two-fold molar excess of the duplex oligodeoxynucleotide substrate 5′d- GGACTCC CCTGAGG.

Original languageEnglish (US)
Pages (from-to)15-21
Number of pages7
JournalGene
Volume74
Issue number1
DOIs
StatePublished - Dec 25 1988

Keywords

  • DNA methylation
  • Recombinant DNA
  • nucleotide sequencing
  • protein crystals
  • tac promoter

ASJC Scopus subject areas

  • Genetics

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