The cloning, purification and characterization of the Eco RV modification methylase.
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, pVIC1, 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.
Nwosu
V U
VU
Department of Biochemistry, University of Southampton, UK.
Connolly
B A
BA
Halford
S E
SE
Garnett
J
J
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
C056127:DNA modification methylase EcoRV; D008780:Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes