Purification and characterization of the MβRsrI DNA methyltransferase from Escherichia coli》.pdfVIP

Ge,le, 118 (1992) 5-l 1 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119~92~505.00 5 GENE 06596 Purification and characterization of the M l RsrI DNA methyltransferase from Escherichia coli (DNA mcthylation; fluorescence spectroscopy; velocity sedimentation; sinefungin) Wiweka Kaszubska”, Heather K. Webb* and Richard I. Gumport Received by F. Barany: IO Dccembcr 1991; Accepted: 23 January 1992: Resubmitted 30 April 1992; Rcccivcd at publishers: I2 allay 1992 SUMMARY The gene (rsrlM) encoding the RsvI DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides was cloned and expressed in Escherichia coli. Under the control of a bacteriophage T7 promoter, 2 O0 of the total protein in a crude extract was M.RsvI. This level of expression represents an approximately 50-fold increase over that present in the natural host. Chromatography using DNA cellulose and the S-adenosylmethionine analogue, sinefungin, was useful in purifying the enzyme to homogeneity. The purification yielded 100 times more enzyme than was obtained from the same quantity of R. xphaeroides cell paste. M.RsvI deposits one methyl group per productive DNA-binding event, as does its functional but sequence-nonhomologous analogue, M.EcoRI. Unlike M.EcoRI, the R. sphaeroides enzyme i