Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70.
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.
Matambo
Tonderayi S
TS
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa.
Odunuga
Odutayo O
OO
Boshoff
Aileen
A
Blatch
Gregory L
GL
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
D003470:Culture Media; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D006632:Histamine; D000251:Adenosine Triphosphatases