Production and purification of refolded recombinant Plasmodium falciparum β-ketoacyl-ACP reductase from inclusion bodies

Karmodiya, Krishanpal ; Kumar Srivastav, Ratnesh ; Surolia, Namita (2005) Production and purification of refolded recombinant Plasmodium falciparum β-ketoacyl-ACP reductase from inclusion bodies Protein Expression and Purification, 42 (1). pp. 131-136. ISSN 1046-5928

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Official URL: http://www.sciencedirect.com/science/article/pii/S...

Related URL: http://dx.doi.org/10.1016/j.pep.2005.02.008

Abstract

A recombinant form of Plasmodium falciparum β -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 Ni2+ 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 μg/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° 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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Plasmodium falciparum; Malaria; Fatty Acid Biosynthesis; β-Ketoacyl-ACP Reductase; Acyl Carrier Protein
ID Code:61601
Deposited On:15 Sep 2011 12:45
Last Modified:15 Sep 2011 12:45

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