Overexpression of BsoBI restriction endonuclease in E. 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 stearothermophilus 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 generate a four-base 5' extension. The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695-699]. Here we report the overexpression of BsoBI restriction endonuclease gene in E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive 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 and transferred into E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, 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.
Ruan
H
H
New England Biolabs Inc., Beverly, MA 01915, USA.
Lunnen
K D
KD
Pelletier
J J
JJ
Xu
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D014644:Genetic Variation; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific