Purification, Properties and Determinations of Recognition Sequence and Cleavage Site of Restriction Endonuclease from “Agrobacterium gelatinovorum”IAM 12617, a Marine Bacterium (AgeI)

Abstract

A new restriction endonuclease, designated as AgeI, was purified from cell-free extracts of a marine bacterium, “Agrobacterium gelatinovorum” IAM 12617 by streptomycin treatment, ammonium sulfate fractionation, combined column Chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q (HR 5/5) and Superose 12 (HR 10/30). The purified enzyme was homogeneous on SDS-polyacrylamide gel disc electrophoresis and free from other phosphatase and exonuclease activities on ligation-recutting test. The relative molecular mass of the enzyme was 24,000 daltons by SDS-polyacrylamide gel disc electrophoresis. The gel filtration using Superose 12 (HR 10/30) gave the same calculation (23,000 daltons). These data indicated that the enzyme is a monomer. The isoelectric point of the enzyme was 6.5. The purified enzyme cleaved λ and Ad2 DNAs at 10 or more and 5 sites, respectively. However, the purified enzyme did not cleave SV40, ϕX174 RF I, M13mpl8 RF I or pBR322 DNAs. pBR328 DNA was cleaved at 1 site by the purified enzyme. The purified enzyme worked best at 37°C and pH 7.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10mm Tris–HCl, 7mm 2-mercaptoethanol, 7mm MgCl2 and 50 mm NaCl. The purified enzyme did not require monovalent cations necessarily for the enzyme reaction. The enzyme recognized the palindromic hexanucleotide DNA sequence 5′-ACCGGT-3′ and cut between A and C, producing a 5′-cohesive tetranucleotide extension.