Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.
A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.
Velkov
Tony
T
Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. tony.velkov@vcp.monash.edu.au
Jones
Alun
A
Lim
Maria L R
ML
D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D007763:Lac Operon; D013058:Mass Spectrometry; D009532:Nickel / Q000737:chemistry*
D009532:Nickel