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

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. similar to 30-40 mg pure recombinant enzyme with a specific activity of 12.6 units/mg could be obtained from a liter of Escherichia coli culture. This enzyme is a homotetramer with an optimal pH similar to 9. Kinetic measurements gave K-mNAD = 0.28 +/- 0.3 mM and K-mG3p = 0.25 +/- 0.03 mM. 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 similar to 27, similar to 37 and similar to 51 kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the similar to 51 kDa 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 membranes) examined had Gapdh associated with them. Each fraction had multiple molecular species associated with them. Such species could arise only by multiple post-translational modifications. Structural heterogeneity observed among molecular species of PyGapdh and their diverse subcellular distribution, supports the view that Gapdh is likely to have multiple nonglycolytic functions in the parasite and could be an effective target for anti-malarial chemotherapeutics. (C) 2015 Elsevier Inc. All rights reserved.