A rapid purification procedure for the HsdM protein of EcoR124I and biophysical characterization of the purified protein

James E. N. Taylor, Anna Swiderska, G. Geoff Kneale

Research output: Contribution to journalArticlepeer-review

Abstract

Type I restriction–modification (R–M) systems are comprised of two multi-subunit enzymes with complementary functions: the methyltransferase (∼160 kDa), responsible for methylation of DNA, and the restriction endonuclease (∼400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits, including HsdM. Characterisation of either enzyme first requires the expression and purification of its constituent subunits, before reconstitution of the multisubunit complex. Previously, purification of the HsdM protein had proved problematic, due to the length of time required for the purification and its susceptibility to degradation. A new protocol was therefore developed to decrease the length of time required to purify the HsdM protein and thus prevent degradation. Finally, we show that the HsdM subunit exhibits a concentration dependent monomer–dimer equilibrium.
Original languageEnglish
Pages (from-to)136-140
Number of pages5
JournalProtein Expression and Purification
Volume87
Issue number2
Early online date27 Nov 2012
DOIs
Publication statusPublished - 1 Feb 2013

Keywords

  • Type I restriction enzymes
  • DNA methyltransferases
  • analytical ultracentrifugation

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