Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus

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Abstract
The restriction endonuclease BstI was purified from 70 kg of B. stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit MW of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7-9.5, and in the presence of 0.5-2 mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for B. amyloliquefaciens), i.e.: .**GRAPHIC**. In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: .**GRAPHIC**.