Cloning, expression, purification and characterization of Plasmodium spp. glyceraldehyde-3-phosphate dehydrogenase

Protein Expression and Purification
Prakash B SangolgiGotam K Jarori

Abstract

Plasmodium spp. solely rely on glycolysis for their energy needs during asexual multiplication in human RBCs, making the enzymes of this pathway potential drug targets. We have cloned, over-expressed and purified Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGapdh) for its kinetic and structural characterization. ∼30-40mg pure recombinant enzyme with a specific activity of 12.6units/mg could be obtained from a liter of Escherichia coli culture. This enzyme is a homotetramer with an optimal pH∼9. Kinetic measurements gave KmNAD=0.28±0.3mM and KmG3P=0.25±0.03mM. Polyclonal antibodies raised in mice showed high specificity as was evident from their non-reactivity to rabbit muscle Gapdh. Western blot of Plasmodium yoelii cell extract showed three bands at MW ∼27, ∼37 and ∼51kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the ∼51kDa form was present only in the soluble fraction of the extract. Subcellular distribution of Gapdh in P. yoelii was examined using differential detergent fractionation method. Each fraction was analyzed on a two-dimensional gel and visualized by Western blotting. All four subcellular fractions (i.e., cytosol, nucleus, cytoskeleton and cell membr...Continue Reading

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Citations

Aug 24, 2016·The Journal of Experimental Medicine·Sung-Jae ChaMarcelo Jacobs-Lorena

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