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 K(m) of 2.0 microM and a turnover number, k(cat), of 0.036s(-1) using the internally quenched fluorescent synthetic peptide substrate EDANS-CO-CH(2)-CH(2)-CO-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-Dap-(DABCYL)-OH (2837b) at pH 5.2.
Xiao
Huogen
H
Department of Food Science, University of Guelph, ON, Canada.
Briere
Lee-Ann K
LA
Dunn
Stanley D
SD
Yada
Rickey Y
RY
D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019281:Dimerization; D007700:Kinetics; D010963:Plasmodium falciparum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D057055:Aspartic Acid Proteases