Plasmodium falciparum:: Enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 degrees C with 0.5 mM isopropyl P-D-thiogalactoside (lPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 degrees C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 mu mol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k(cat) [(3.2 +/- 0.02) x 10(4)] with 3-acetylpyridine adenine dinucleotide (APAD(+)). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH. (C) 2008 Elsevier Inc. All rights reserved.