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Expression and characterisation of Plasmepsin I from Plasmodium falciparum

Moon, Richard P., Tyas, Lorraine, Certa, Ulrich, Rupp, Katharina, Bur, Daniel, Jacquet, Catherine, Matile, Hugues, Loetscher, Hansruedi, Grueninger-Leitch, Fiona, Kay, John, Dunn, Ben M., Berry, Colin ORCID: and Ridley, Robert G. 1997. Expression and characterisation of Plasmepsin I from Plasmodium falciparum. European Journal of Biochemistry 244 (2) , pp. 552-560. 10.1111/j.1432-1033.1997.00552.x

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Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5–10–fold lower Kcat/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Subjects: Q Science > QR Microbiology
Additional Information: malaria; Plasmodium falciparum; plasmepsin I; aspartic protease
Publisher: Wiley-Blackwell
ISSN: 0014-2956
Last Modified: 27 Oct 2022 09:13

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