ISOLATION OF A STRAIN OVERPRODUCING ENDONUCLEASE ECO29KI - PURIFICATION AND CHARACTERIZATION OF THE HOMOGENEOUS ENZYME

The physical map of the plasmid pSACII1 carrying the genes of restrict ion-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 R8 02 [pECO29A15] with Eco29kI synthesis level about 100 times higher tha n in the parent strain. The restriction endonuclease was purified from Escherichia coli K802 [pECO29A15] cells to near homogeneity using col umn chromatography sequentially on phosphocellulose, hydroxyapatite, a nd heparin-Sepharose and rechromatography on phosphocellulose. Biochem ical characterization of the homogeneous R-Eco29kI is given. The enzym e has molecular mass 24.5 kD and is present in the solution as a monom er.