Isolation and purification of 3-oxo-Δ5-steroid isomerase from crude E-coli lysate by countercurrent chromatography

KSI (3-oxo-Delta(5)-steroid isomerase) was purified from crude E. coli lysate by countercurrent chromatography using a polymer phase system composed of polyethylene glycol 3350 and potassium phosphate (pH 7), each at 12.5% (w/w) in distilled water. Using the cross-axis coil planet centrifuge, a preparative scale separation was performed on 25 mL E. coli lysate containing ca 50 mg of KSI. About 40 mg of KSI was recovered at 98% purity. A small scale separation was performed on ca. 3 mg of N-15-labeled KSI using the toroidal coil centrifuge. The present method eliminates sample loss and denaturation caused by the solid support and yields pure proteins in both preparative and analytical separations.