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Isolation and Purification of 3-Oxo-Δ 5 -Steroid Isomerase from Crude E. coli Lysate by Countercurrent Chromatography

1998; Taylor & Francis; Volume: 21; Issue: 1-2 Linguagem: Inglês

10.1080/10826079808001937

1520-572X

Lin Qi, Ying Ma, Yoichiro Ito, Henry M. Fales,

Natural product bioactivities and synthesis

Abstract KSI (3-oxo-Δ5-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 25mL 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. 3mg of 15N-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.