Figure 1

(A) Native and recombinant expression of PfHsp90: E. coli BL21(DE3) containing pMICO plasmid [32] and either the pET23a-PfHsp90 clone (lane C) or just the pET-23a vector (no insert) (lane V) were induced with IPTG, analyzed in SDS-PAGE, followed by staining. Proteins from duplicate gels were probed in immunoblot with antibody against T7-tag or Hsp90 (Sigma), as described under Materials and Methods. In the far right panel, 50 μg of total parasitic protein was similarly analyzed, and probed with the same anti-Hsp90 antibody. The roughly 92 k His-tagged recombinant and 90 k parasitic Hsp90 bands are indicated by arrowheads (left and right, respectively); Mr values (in thousands) of protein markers are shown on the left. (B) Stage-specific expression of Hsp90 mRNA in the parasite: Parasites were synchronized by sorbitol treatment and harvested at time points corresponding to the following stages: 20 h (early trophozoite), 26 h (late trophozoite), 32 h (early schizont), 40 h (late schizont), 48 h (ring, from the second cycle of synchrony). The upper panel shows a Northern of the total parasitic RNA probed with ³²P-labeled PCR product corresponding to nucleotide 450–990 of Hsp90 ORF. The 2.9 kb Hsp90 mRNA is so indicated. The lower panel is an ethidium bromide stained picture of the parental gel, showing the ribosomal RNAs and size markers (lane M). (C) Agreement of Northern data with microarray analysis [34]. The intensities of the PfHsp90 mRNA bands (panel B) were quantified by densitometry, normalized against rRNA, and plotted as green bars of relative height. The bars were superimposed on the DeRisi microarray data for Hsp90 mRNA throughout the intraerythrocytic cycle [34]. (D) Stage-specific expression of the Hsp90 protein: Immunoblot of 30 μg of total protein from the same parasitic stages as in panel (B). The Immunoblot for PfARP protein, which is constitutively expressed in all stages [35], serves as control for sample loading.