Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.
The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/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 mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). 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: (formula: see text).
D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004247:DNA; D004587:Electrophoresis, Agar Gel; D004590:Electrophoresis, Paper; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008970:Molecular Weight
D004247:DNA; D004720:Endonucleases