Expression and enzymatic characterization of the soluble recombinant plasmepsin I from Plasmodium falciparum.
The plasmepsins are involved in the degradation of host cell hemoglobin during malaria infection. Plasmepsin I (PM I) initiates the degradative process, and has been suggested as an attractive target for the treatment of malaria. The production of active recombinant PM I, however, has been challenging. We report for the first time, the expression and partial characterization of soluble recombinant PM I from Plasmodium falciparum in which a truncated form of PM I (Lys77P-Leu329) (P indicates a propart residues) was fused to thioredoxin in the pET32b(+) vector, Trx-tPM I and expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The soluble fusion protein was purified from cell culture using a combination of Ni(2+) affinity and gel filtration chromatography and was capable of autocatalytic activation at pH 4.0-5.5, which occurred at Leu116P-Ser117P, seven residues upstream of the native cleavage site (Gly123P-Asn1). The mature tPM I (mtPM I) was capable of hydrolyzing both human hemoglobin with a pH optimum of pH 2.8-4.0 and the synthetic fluorogenic peptide EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH with a dual pH optima of pH 2.5-3.0 and pH 4.5-5.5. Using the synthetic substrate, mtPM I exhibited kinetic parameters comparable to native PM I.
Xiao
Huogen
H
Department of Food Science, University of Guelph, Guelph, ON, Canada N1G 2W1.
Tanaka
Takuji
T
Ogawa
Masahiro
M
Yada
Rickey Y
RY
D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry*; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry*; D001665:Binding Sites; D005786:Gene Expression Regulation; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010963:Plasmodium falciparum / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry
D000953:Antigens, Protozoan; D006454:Hemoglobins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin