Matambo, T, Odunuga, O, Boshoff, Aileen and Blatch, Gregory ORCID: 0000-0003-0778-8577 (2004) Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70. Protein Expression and Purification, 33 (2). pp. 214-222. ISSN 1046-5928

Abstract

Plasmodium falciparum heat shock protein (PfHsp70) has been proposed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. However, the biochemical and chaperone properties of PfHsp70 have not been elucidated. The heterologous overproduction of P. falciparum proteins in Escherichia coli is problematic because of its AT-rich genome and the usage of codons that are rarely used in E. coli. In this paper, we describe the successful overproduction of (His)6- PfHsp70 in E. coli using the pQE30 expression vector system. Initial experiments with E. coli [pQE30/PfHsp70] resulted in the overproduction of the full-length protein and truncated derivatives. The RIG plasmid, which encodes tRNAs for rare codons, was engineered into the E. coli [pQE30/PfHsp70] strain, resulting in significant reduction of the truncated (His)6-PfHsp70 derivatives and improved yields of the full-length protein. (His)6-PfHsp70 was successfully purified using nickel-chelating Sepharose affinity chromatography and its biochemical properties were determined. The Vmax, Km, and kcat for the basal ATPase activity of (His)6- PfHsp70 were found to be 14.6 nmol/min/mg, 616.5 lM, and 1.03 min�1, respectively. Gel filtration studies indicated that (His)6- PfHsp70 existed largely as a monomer in solution. This is the first study to biochemically describe PfHsp70 and establishes a foundation for future studies on its chaperone properties.

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