title: Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70.
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 V(max), K(m), and k(cat) for the basal ATPase activity of (His)(6)-PfHsp70 were found to be 14.6 nmol/min/mg, 616.5 microM, 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.
authors.affiliation: Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa.
authors.forename: Tonderayi S
authors.initials: TS
authors.lastname: Matambo
authors.forename: Odutayo O
authors.initials: OO
authors.lastname: Odunuga
authors.forename: Aileen
authors.initials: A
authors.lastname: Boshoff
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Blatch
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006632:Histamine / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D010963:Plasmodium falciparum / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014169:Transformation, Bacterial
chemical_list: D003470:Culture Media; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D006632:Histamine; D000251:Adenosine Triphosphatases