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Nucleic Acids Research, 1988, Vol. 16, No. 9 3705-3720
© 1988

Articles

The cloning, purification and characterization of the Eco RV modification methylase

Victor U. Nwosu, Bernard A. Connolly, Stephen E. Halford1 and Jane Garnett1

Department of Biochemistry, University of Southampton Southampton, SO9 3TU UK 1Department of Biochemistry, University of Bristol Bristol, BS8 ITD, UK

Received February 9, 1988. Revised March 28, 1988. Accepted March 28, 1988.

The gene for the Eco RV methylase has been cloned into a plasmid under control of the strong {lambda}PL promoter and overexpressed in E. coli. This plasmid, pVICl, gives reliable overexpression of the methylase at levels of about 20% of total protein. Maximum yields of soluble protein are achieved after about 6 hours of induction. If the cells are harvested later than this much of the enzyme is found in the pellet fraction following centrifugation. A two column purification scheme using phosphocellulose and Blue-Sepharose chromatography has been developed. This yielded pure methylase in amounts of 5mg per gram E. coli cell paste. The enzyme is monomeric and methylates the first deoxyadenosine residue in its recognition sequence GATATC.


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