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. Solonin^1,3

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.

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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.

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KEY WORDS: plasmid, site-specific endonuclease, restriction-modification, Escherichia coli.

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