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 of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried out. High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncated profalcipain-2 construct. Recombinant falcipain-2 was expressed as inclusion bodies, solubilized, and purified by nickel affinity chromatography. A systematic approach was then used to optimize refolding parameters. This approach utilized 100-fold dilutions of reduced and denatured falcipain-2 into 203 different buffers in a microtiter plate format. Refolding efficiency varied markedly. Optimal refolding was obtained in an alkaline buffer containing glycerol or sucrose and equal concentrations of reduced and oxidized glutathione. After optimization of the expression and refolding protocols and additional purification with anion-exchange chromatography, 12 mg of falcipain-2 was obtained from 5 liters of E. coli, and crystals of the protease were grown. The systematic approach described here allowed the rapid evaluation of a large number of expression and refolding conditions and provided milligram quantities of recombinant falcipain-2.
Sijwali
P S
PS
Department of Medicine, San Francisco General Hospital, San Francisco, CA 94143, USA.
Brinen
L S
LS
Rosenthal
P J
PJ
D000818:Animals; D001120:Arginine / Q000378:metabolism; D003460:Crystallization; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D001120:Arginine; D003546:Cysteine Endopeptidases; C416224:falcipain 2; D005990:Glycerol