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

doi:10.1016/j.pep.2012.11.003

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 journal › Article › peer-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 language	English
Pages (from-to)	136-140
Number of pages	5
Journal	Protein Expression and Purification
Volume	87
Issue number	2
Early online date	27 Nov 2012
DOIs	https://doi.org/10.1016/j.pep.2012.11.003
Publication status	Published - 1 Feb 2013

Keywords

Type I restriction enzymes
DNA methyltransferases
analytical ultracentrifugation

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10.1016/j.pep.2012.11.003

Cite this

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title = "A rapid purification procedure for the HsdM protein of EcoR124I and biophysical characterization of the purified protein",

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.",

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AU - Taylor, James E. N.

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AU - Kneale, G. Geoff

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AB - 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.

KW - Type I restriction enzymes

KW - DNA methyltransferases

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