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 0.5mM isopropyl beta-d-thiogalactoside (IPTG) 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 micromol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k(cat) [(3.2+/-0.02)x10(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.
Berwal
Ritu
R
Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, Madhya Pradesh, India.
Gopalan
Natarajan
N
Chandel
Kshitij
K
Prasad
G B K S
GB
Prakash
Shri
S
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D013696:Temperature
D016054:DNA, Protozoan; D007770:L-Lactate Dehydrogenase