OVEREXPRESSION OF BSOBI RESTRICTION-ENDONUCLEASE IN ESCHERICHIA-COLI,PURIFICATION OF THE RECOMBINANT BSOBI, AND IDENTIFICATION OF CATALYTIC RESIDUES OF BSOBI BY RANDOM MUTAGENESIS

BsoBI is a type II restriction enzyme found in Bacillus stearothermoph ilus JN209 that recognizes the symmetric sequence 5'-CYCGRG-3' (Y=C or T; R=A or G) and cleaves between the first and second base to generat e a four-base 5' extension. The cloning and sequencing of BsoBI restri ction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695-699]. Here we report the overexpression of BsoB I restriction endonuclease gene in E. coli by insertion of the endonuc lease gene into an expression vector pRRS. The recombinant BsoBI was p urified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitu tive expression of BsoBI from pRRS is lethal to E. coli in the absence of the cognate methylase. The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro an d transferred into E. coli via plasmid vectors in the absence of the c ognate methylase. Surviving transformants were selected that carry Bso BI variants which lost endonuclease activity. DNA sequencing of the mu tant alleles revealed that G123, D124, D212, D246, E252 and H253 are i mportant residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-defic ient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D 246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.