Skip to content
Back to outputs

A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI

Research output: Contribution to journalArticlepeer-review

Standard

A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI. / Marks, P.; McGeehan, John; Kneale, Geoff.

In: Protein Expression and Purification, Vol. 37, No. 1, 2004, p. 236-242.

Research output: Contribution to journalArticlepeer-review

Harvard

Marks, P, McGeehan, J & Kneale, G 2004, 'A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI', Protein Expression and Purification, vol. 37, no. 1, pp. 236-242. https://doi.org/10.1016/j.pep.2004.06.008

APA

Vancouver

Author

Marks, P. ; McGeehan, John ; Kneale, Geoff. / A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI. In: Protein Expression and Purification. 2004 ; Vol. 37, No. 1. pp. 236-242.

Bibtex

@article{10b28e4771c4468a86462e8ee3f08f07,
title = "A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI",
abstract = "Biochemical and structural studies of the methylase from the type 1 R–M system AhdI require the ability to purify this multi-subunit enzyme in significant quantities in a soluble and active form. Several Escherichia coli expression systems were tested for their ability to produce the intact methylase but this could not be achieved in a simple co-expression system. Expression experiments were optimised to produce high yields of soluble M and S subunits as individual proteins. Temperature and conditions of induction proved to be the most useful factors and although purification of the S subunit was successful, an efficient strategy for the M subunit remained elusive. A novel strategy was developed in which individual subunits are expressed separately and the bacterial cells mixed before lysis. This method produced a high yield of the multi-subunit methylase when purified to homogeneity by means of heparin and size-exclusion chromatography. It was found to be essential, however, to remove tightly bound DNA by ammonium sulphate precipitation in 1 M NaCl. The intact methylase can now be consistently produced, avoiding the use of fusion proteins. The purified enzyme is stable over long time periods, unlike the individual subunits. This method may be of general application where the expression of multi-subunit proteins, or indeed their individual components, is problematic.",
author = "P. Marks and John McGeehan and Geoff Kneale",
year = "2004",
doi = "10.1016/j.pep.2004.06.008",
language = "English",
volume = "37",
pages = "236--242",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "1",

}

RIS

TY - JOUR

T1 - A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI

AU - Marks, P.

AU - McGeehan, John

AU - Kneale, Geoff

PY - 2004

Y1 - 2004

N2 - Biochemical and structural studies of the methylase from the type 1 R–M system AhdI require the ability to purify this multi-subunit enzyme in significant quantities in a soluble and active form. Several Escherichia coli expression systems were tested for their ability to produce the intact methylase but this could not be achieved in a simple co-expression system. Expression experiments were optimised to produce high yields of soluble M and S subunits as individual proteins. Temperature and conditions of induction proved to be the most useful factors and although purification of the S subunit was successful, an efficient strategy for the M subunit remained elusive. A novel strategy was developed in which individual subunits are expressed separately and the bacterial cells mixed before lysis. This method produced a high yield of the multi-subunit methylase when purified to homogeneity by means of heparin and size-exclusion chromatography. It was found to be essential, however, to remove tightly bound DNA by ammonium sulphate precipitation in 1 M NaCl. The intact methylase can now be consistently produced, avoiding the use of fusion proteins. The purified enzyme is stable over long time periods, unlike the individual subunits. This method may be of general application where the expression of multi-subunit proteins, or indeed their individual components, is problematic.

AB - Biochemical and structural studies of the methylase from the type 1 R–M system AhdI require the ability to purify this multi-subunit enzyme in significant quantities in a soluble and active form. Several Escherichia coli expression systems were tested for their ability to produce the intact methylase but this could not be achieved in a simple co-expression system. Expression experiments were optimised to produce high yields of soluble M and S subunits as individual proteins. Temperature and conditions of induction proved to be the most useful factors and although purification of the S subunit was successful, an efficient strategy for the M subunit remained elusive. A novel strategy was developed in which individual subunits are expressed separately and the bacterial cells mixed before lysis. This method produced a high yield of the multi-subunit methylase when purified to homogeneity by means of heparin and size-exclusion chromatography. It was found to be essential, however, to remove tightly bound DNA by ammonium sulphate precipitation in 1 M NaCl. The intact methylase can now be consistently produced, avoiding the use of fusion proteins. The purified enzyme is stable over long time periods, unlike the individual subunits. This method may be of general application where the expression of multi-subunit proteins, or indeed their individual components, is problematic.

U2 - 10.1016/j.pep.2004.06.008

DO - 10.1016/j.pep.2004.06.008

M3 - Article

VL - 37

SP - 236

EP - 242

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -

ID: 155137