Ni²⁺-Based Immobilized Metal Ion Affinity Chromatography of Lactose Operon Repressor Protein from Escherichia Coli

Abstract

A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni²⁺-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.

Keywords:

• Immobilized metal ion affinity chromatography
• Lac repressor
• Protein crystallization
• Protein purification

ACKNOWLEDGMENT

T. Velkov is the recipient of a Peter Doherty Fellowship (384300) from the National Health and Medical Research Council, Australia.

Notes

The soluble fraction from the same cell lystate was employed as the starting material for each method.

^a Density of the lac repressor band as a percentage of the total protein density estimated by densitometric analysis of SDS-PAGE gels (cf Fig. 2).

^b Starting protein mass used for the phosphocellulose-based purification coupled with the MonoQ polishing step.

^c Starting protein mass used for the phosphocellulose-based purification coupled with the Sephadex G-200 polishing step.

^d NA, not applicable.