Purification of the HhaII restriction endonuclease from an overproducer Escherichia coli clone.
An Escherichia coli K12 strain carrying the HhaII methylase and restriction genes on two separate compatible plasmids, pSK5 and pSK7, is used to overproduce the restriction endonuclease. Plasmid pSK5 expresses the methylase gene constitutively from its chloramphenicol resistance gene promoter, and plasmid pSK7 expresses the restriction endonuclease under control of the lacUV5 promoter. Induction of the two-plasmid clone with 1 mM isopropyl-1-thio-beta-D-galactopyranoside results in a 15-fold increase in HhaII endonuclease activity. The enzyme has been purified to apparent homogeneity. It migrates as a 23-kilodalton polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide electrophoretic gels and as a 52-kilo-dalton native protein dimer on a high pressure liquid chromatography sizing column.
D002701:Chloramphenicol / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009876:Operon; D010957:Plasmids
D002701:Chloramphenicol; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases