Production and purification of refolded recombinant Plasmodium falciparum beta-ketoacyl-ACP reductase from inclusion bodies.
A recombinant form of Plasmodium falciparum beta-ketoacyl-ACP reductase (PfFabG) was overexpressed in Escherichia coli BL-21 codon plus (DE3). The resulting insoluble inclusion bodies were separated from cellular debris by extensive washing with buffer containing 0.05% Tween 20 and solubilized by homogenization with 8 M urea. Attempts to refold PfFabG from solubilized inclusion bodies employing Rotofor (separation based on different pIs of proteins in a mixture) followed by Ni(2+) or cation exchange chromatography were not successful either by bringing down the urea concentration instantaneously, stepwise, or by dialysis. Denatured PfFabG was therefore initially purified by cation exchange chromatography and was then correctly refolded at a final concentration of 100-200 microg/ml in a 20 mM Na-acetate buffer, pH 5.3, with 300 mM NaCl, 10% glycerol, and 0.05% Tween 20. The protein was found to be properly folded only in the presence of the cofactor NADPH and salt at a concentration 300 mM by drop dilution method at 2-8 degrees C for 12 h. The purified final product was >98% pure by denaturing gel electrophoresis. The purified protein was biologically active in a standard enzymatic assay using acetoacetyl-CoA as a substrate. The enzyme was found to be stable up to fourth day of purification and glycerol was found to stabilize enzyme activity for several weeks, during storage. This effort paves the way for elucidation of the structure-function correlations for PfFabG as well as exploration of the enzyme for developing inhibitors against it for combating malaria.
Karmodiya
Krishanpal
K
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Srivastav
Ratnesh Kumar
RK
Surolia
Namita
N
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism*; D007700:Kinetics; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase