Co-expression of the Plasmodium falciparum molecular chaperone, PfHsp70, improves the heterologous production of the antimalarial drug target GTP cyclohydrolase I, PfGCHI.
Molecular chaperones have been used for the improved expression of target proteins within heterologous systems; however, the chaperone and target protein have seldom been matched in terms of origin. We have developed a heterologous co-expression system that allows independent expression of the plasmodial chaperone, PfHsp70, and a plasmodial target protein. In this study, the target was Plasmodium falciparum GTP cyclohydrolase I (PfGCHI), the first enzyme in the plasmodial folate pathway. The sequential expression of the molecular chaperone followed by the target protein increased the expression of soluble functional PfGCHI. His-tagged PfGCHI was successfully purified using nickel affinity chromatography, and the specific activity was determined by high performance liquid chromatography with spectrofluorometeric detection to be 5.93nmol/h/mg. This is the first report of a heterologous co-expression system in which a plasmodial chaperone is harnessed for the improved production and purification of a plasmodial target protein.
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, South Africa.
Linda L
LL
Stephens
Addmore
A
Shonhai
Gregory L
GL
Blatch
D000962:Antimalarials / Q000627:therapeutic use; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D006136:GTP Cyclohydrolase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000469:parasitology; D058990:Molecular Targeted Therapy; D009842:Oligopeptides / Q000378:metabolism; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence
D000962:Antimalarials; D018840:HSP70 Heat-Shock Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006639:Histidine; D006136:GTP Cyclohydrolase