Large-scale purification of two forms of active lac operator from plasmids.
A practical procedure is described for obtaining milligram quantities of a small (29 nucleotide) Eco RI restriction fragment of DNA containing the Escherichia coli lac operator. A yield of 10--15 mg of operator is obtained from 1 kg of wet cell paste. The resultant operator is shown to be homogeneous and competitively active in filter assays. Two separable but interconvertible forms of lac operator exist in solution, probably linear duplex and hairpin isomers. Only the presumed linear form is active in binding lac repressor by competition assay, but the two isomers are interconvertible by heating to 80 degrees C. The methods described here should be generally applicable for purifying other restriction fragments from plasmids.
D002850:Chromatography, Gel; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000032:analysis; D007763:Lac Operon; D008722:Methods; D010636:Phenols; D010957:Plasmids; D011402:Pronase / Q000494:pharmacology; D012260:Ribonucleases / Q000494:pharmacology
D004269:DNA, Bacterial; D010636:Phenols; D012260:Ribonucleases; D004262:DNA Restriction Enzymes; D011402:Pronase