High-level expression in Escherichia coli, purification and kinetic characterization of Plasmodium falciparum M1-aminopeptidase

• We optimized and cloned in pTrcHis2B vector a PfAM1 gene for bacterial expression.
• We obtained 100 mg/L soluble rPfAM1 by 18 h-IPTG induction at 37 °C in E. coli BL21.
• N-terminal His-tagged rPfAM1 was purified 27-fold in a single step by IMAC.
• rPfAM1 has optimal pH, substrate specificity and inhibition profile as PfAM1.
• Our strategy allows producing sufficient enzyme to screen for PfAM1 inhibitors.

Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo proteases, is a promising target for malaria, a devastating human parasitic disease. We report the high-level expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector. Optimal expression was achieved by induction with 1 mM IPTG at 37 °C for 18 h. This allowed obtaining 100 mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass was from rPFAM1. rPfAM1, fused to an amino-terminal 6×His tag, was purified in a single step by immobilized metal ion affinity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition profile typical of metallo-aminopeptidases, and inhibition from Zn2+ excess, were similar to those of the native PfAM1. We have thus optimized an expression system that should be useful for identifying new PfAM1 inhibitors.