Yield improvement and enzymatic dissection of Plasmodium falciparum plasmepsin V

In this study, we employed a multipurpose fusion tag to improve the production of PfPMV in E. coli. Recombinant PfPMVm, comprising residues 84 to 521, was substantially obtained in soluble form and could be purified in a single step, yielding a 3.7-fold increase in purified PfPMVm compared to previous reports. Additionally, we have mutated the catalytic residues (D118 N and D365 N), individually and together, and the unpaired cysteine residue C178 to evaluate the effects on catalytic efficiency. Mutation of D365 had more pronounced effects on the catalytic efficiency than that of D118, suggesting that the D365 may act as a catalytic nucleophile to activate the water molecule. The importance of C178 was also confirmed by the inhibition by metal ions, indicating that C178 is partially involved in the substrate recognition. Collectively, our results describe an improved system to produce recombinant PfPMVm in E. coli and dissect the amino acids involved in catalysis and substrate recognition.
Source: Molecular and Biochemical Parasitology - Category: Parasitology Source Type: research