ABSTRACT
A simple and fast method for isolation and purification of SsoI methylase from the bacterial strain Shigella sonnei 47 has been proposed. The enzyme is a modifying component of host cell specificity system and protects the acceptor DNA from hydrolysis by restriction endonucleases SsoI and EcoRI. The method is based on the hydrophobic chromatography of ammonium sulphate fraction on the phenylsepharose. The enzyme preparation obtained is devoid of specific and nonspecific endonucleases traces and is stable at storage in 30% glycerol during a year. The conditions of manifestation of "stroke" activity by the enzyme were studied.
Subject(s)
Shigella sonnei/enzymology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/isolation & purification , Base Sequence , Hydrogen-Ion Concentration , Isoelectric FocusingABSTRACT
Shigella sonnei 47 cells were found to contain DNA-methylase SsoII which is a modifying component of the system of host specificity of SsoII. The recognition sequence (RS) of methylase SsoII is represented by a five-member palyndromic structure--5'...CCNGG...3'--with a degenerated central nucleotide. Modification of SsoII affords protection of acceptor DNA not only from SsoII type restriction, but also from other restrictases, e. g., Eco RII having an analogous RS but with a less degenerated central nucleotide pair. A simple and rapid procedure for isolation and purification of DNA-methylase ScoII, which employs hydrophobic chromatography on phenyl-Sepharose, has been developed. The enzyme preparation does not contain trace amounts of specific and nonspecific endonucleases and keeps stable on storage in 30% glycerol over a period of one year.
