title: Isolation of a strain overproducing endonuclease Eco29kI: purification and characterization of the homogeneous enzyme.
abstract: The physical map of the plasmid pSACII1 carrying the genes of restriction-modification system Eco29kI (isoschizomer of SacII) was determined. The cloning of the Eco29kI endonuclease and methylase genes into the plasmid vector pUC129 produced recombinant strain Escherichia coli K802[pECO29A15] with Eco29kI synthesis level about 100 times higher than in the parent strain. The restriction endonuclease was purified from Escherichia coli K802 [pECO29A15] cells to near homogeneity using column chromatography sequentially on phosphocellulose, hydroxyapatite, and heparin-Sepharose and rechromatography on phosphocellulose. Biochemical characterization of the homogeneous R Eco29kI is given. The enzyme has molecular mass 24.5 kD and is present in the solution as a monomer.
authors.affiliation: Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Moscow Region, Russia.
authors.forename: A V
authors.initials: AV
authors.lastname: Pertzev
authors.forename: A N
authors.initials: AN
authors.lastname: Kravetz
authors.forename: S G
authors.initials: SG
authors.lastname: Mayorov
authors.forename: M V
authors.initials: MV
authors.lastname: Zakharova
authors.forename: A S
authors.initials: AS
authors.lastname: Solonin
mesh_terms: D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D015183:Restriction Mapping
chemical_list: C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific