Characterization of the monomer–dimer equilibrium of recombinant histo-aspartic protease from Plasmodium falciparum

Histo-aspartic protease (HAP) from Plasmodium falciparum is an intriguing aspartic protease due to its unique structure. Our previous study reported the first recombinant expression of soluble HAP, in its truncated form (lys77p-Leu328) (p denotes prosegment), as a thioredoxin (Trx) fusion protein Trx–tHAP. The present study found that the recombinant Trx–tHAP fusion protein aggregated during purification which could be prevented through the addition of 0.2% CHAPS. Trx–tHAP fusion protein was processed into a mature form of tHAP (mtHAP) by both autoactivation, and activation with either enterokinase or plasmepsin II. Using gel filtration chromatography as well as sedimentation velocity and equilibrium ultracentrifugation, it was shown that the recombinant mtHAP exists in a dynamic monomer–dimer equilibrium with an increasing dissociation constant in the presence of CHAPS. Enzymatic activity data indicated that HAP was most active as a monomer. The dominant monomeric form showed a Km of 2.0 μM and a turnover number, kcat, of 0.036 s−1 using the internally quenched fluorescent synthetic peptide substrate EDANS–CO–CH2–CH2–CO–Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro–Dap–(DABCYL)–OH (2837b) at pH 5.2.

We report for the first time that the recombinant histo-aspartic protease (HAP), an enzyme critical to malaria infection, exists in a dynamic monomer–dimer equilibrium and that the monomeric form is the most active.Figure optionsDownload high-quality image (57 K)Download as PowerPoint slide