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PEDS Advance Access published online on December 10, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm066
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Expression and enzymatic characterizationof the soluble recombinant plasmepsin Ifrom Plasmodium falciparum

Huogen Xiao1, Takuji Tanaka2, Masahiro Ogawa1,3 and Rickey Y. Yada1,4

1Department of Food Science, University of Guelph, Guelph, ON, Canada N1G 2W1 2Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8 3Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan

4 To whom correspondence should be addressed.E-mail: ryada{at}uoguelph.ca

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 Ni2+ 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-CH2-CH2-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.

Keywords: aspartic protease/malaria/plasmepsin I/recombinant expression

Received January 28, 2007; revised October 16, 2007; accepted October 23, 2007.


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