title: Recombinant expression and partial characterization of an active soluble histo-aspartic protease from Plasmodium falciparum.
abstract: Malaria aspartic proteases are attractive drug targets for the treatment of malaria, however, recombinant expression of active histo-aspartic proteinase (HAP) to facilitate its characterization has proven elusive. The present study reports on the first recombinant expression of soluble, active histo-aspartic proteinase from Plasmodium falciparum as a thioredoxin fusion protein. A truncated form of HAP (77p-451) was fused to thioredoxin in the pET32b(+) vector and the fusion protein (Trx-tHAP) was expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The fusion protein was partially purified from the culture medium using a combination of anion exchange and Ni(2+) affinity chromatography. Soluble tHAP was subsequently purified by enterokinase treatment and removal, followed by gel filtration chromatography. Although truncated HAP was incapable of autocatalytic activation, enterokinase digestion of partially purified fusion protein released the truncated prosegment yielding a mature form of tHAP (mtHAP). N-terminal sequencing of mtHAP indicated that enterokinase cleavage took place at Lys119-Ser120, four residues upstream of the native cleavage site (Gly123-Ser124). Initial activity tests showed that mtHAP was capable of hydrolyzing acid-denatured globin as well as cleavage of the synthetic substrate EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH. Inhibition studies showed that the activity of mtHAP was completely inhibited by pepstatin A and to a lesser degree, PMSF. Using the synthetic substrate, mtHAP showed a pH optimum of 5.2, and Km=3.4 microM and kcat=1.6 x 10(-3)s(-1). The successful expression of active recombinant HAP from E. coli will accelerate the investigation of the structure-function relationships of HAP and facilitate the development of specific inhibitors with antimalarial activities.
authors.affiliation: Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1.
authors.forename: Huogen
authors.initials: H
authors.lastname: Xiao
authors.forename: Andras F
authors.initials: AF
authors.lastname: Sinkovits
authors.forename: Brian C
authors.initials: BC
authors.lastname: Bryksa
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ogawa
authors.forename: Rickey Y
authors.initials: RY
authors.lastname: Yada
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D015870:Gene Expression; D005914:Globins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D005914:Globins; D016282:Aspartic Acid Endopeptidases