Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2

The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy. In order to obtain large quantities o f active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the pr otease was carried out. High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncate d profalcipain-2 construct. Recombinant falcipain-2 was expressed as inclus ion bodies, solubilized, and purified by nickel affinity chromatography. A systematic approach was then used to optimize refolding parameters. This ap proach utilized 100-fold dilutions of reduced and denatured falcipain-2 int o 203 different buffers in a microtiter plate format. Refolding efficiency varied markedly. Optimal refolding was obtained in an alkaline buffer conta ining glycerol or sucrose and equal concentrations of reduced and oxidized glutathione. After optimization of the expression and refolding protocols a nd additional purification with anion-exchange chromatography, 12 mg of fal cipain-2 was obtained from 5 liters of E. coli, and crystals of the proteas e were grown. The systematic approach described here allowed the rapid eval uation of a large number of expression and refolding conditions and provide d milligram quantities of recombinant falcipain-2. (C) 2001 Academic Press.