A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI.
Biochemical and structural studies of the methylase from the type 1 1/2 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.
Marks
Phil
P
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth POI 2DT, UK.
McGeehan
John
J
Kneale
Geoff
G
D016980:Aeromonas hydrophila / Q000201:enzymology; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D009097:Multienzyme Complexes; D021122:Protein Subunits; D015254:DNA Modification Methylases