RESUMEN
A specific type II restriction endonuclease T.Smu451I has been purified to electrophoretic homogeneity from the frozen cells of soil bacterium Sphingobacterium multivorum 451 (formerly Flavobacterium multivorum 451), using ultrasonic grinding, nucleic acid removal by streptomycin sulfate, protein precipitation by ammonium sulfate and phosphocellulose P-11, DEAE-Cellulose DE-52, Hepharin-Sepharose CL-6B chromatography, and elucidated several characteristics of T.Smu451I. The molecular weight of the enzyme determined by gel filtration and SDS-polyacrylamide gel electrophoresis was calculated to be 45,000 ± 2000 D (dimer) and 23,000 ± 1000 D (monomer), respectively. The isoelectric point (pI) of T.Smu451I is 5.4. T.Smu451I recognizes pentanucleotide palindromic sequences 5'-GGNC↓C-3' and cleaves between C and C in position shown by arrow to produce 3'-cohesive terminus of trinucleotide. Therefore, T.Smu451I is a neoschizomer of T.AsuI.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II , Sphingobacterium , Cromatografía en Gel , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/aislamiento & purificación , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Sphingobacterium/enzimología , Especificidad por SustratoRESUMEN
Bacillus circulans 528 produces a restriction endonuclease, Bci528I which is an isoschizomer of EcoRI. We purified the enzyme, using Sephadex G-150, Phospho-cellulose, DEAE-cellulose, Hepharin-Sepharose CL-6B chromatography. The specific activity of Bci528I was 29,400 U/mg·protein. Bci528I recognizes 5'-GAATTC-3' in dsDNA and cleaves between G and A of the recognition sequence, producing a symmetric four base 5'overhang.