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Isolation of a Strain Overproducing Endonuclease Eco29kI: Purification and Characterization of the Homogeneous Enzyme

A. V. Pertzev,1 A. N. Kravetz,2 S. G. Mayorov,1 M. V. Zakharova,1 and A. S. Solonin1,3

1Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, Pushchino, Moscow Region, 142292 Russia; fax: (095) 923-36-02; E-mail: solonin@ibpm.serpukhov.su

2Gromashevsky Kiev Research Institute of Epidemiology and Infectious Diseases, Ministry of Health Protection, Protasiv Yar Uzviz 4, Kiev, 252038 Ukraine; E-mail: kravetz@ieid.freenet.kiev.ua

3To whom correspondence should be addressed.

Submitted January 30, 1997; revision submitted April 17, 1997.
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 are given. The enzyme has molecular mass 24.5 kD and is present in the solution as a monomer.
KEY WORDS: plasmid, site-specific endonuclease, restriction-modification, Escherichia coli.