194 Size exclusion chromatography Size exclusion chromatography was conducted as previously described (Pesce et al. 2008). Infected erythrocytes were suspended in PBS (pH 7.4) containing 0.15% (w/v) saponin for 10 min on ice followed by centrifugation at 5,000g for 5 min. The pellet fraction of the parasite was washed three times with PBS (pH 7.4) followed by resuspension in 50 mM Tris, 150 mM NaCl (pH 7.4) including 1 mM PMSF and protease inhibitor cocktail (Pierce) and three freeze–thaw cycles. The parasite lysate was centrifuged at 20,000g for 20 min at 4°C to separate soluble parasite lysate and cell debris. An aliquot containing 400 μg of total parasite lysate was injected into a 24-ml Superdex 200 column (GE Healthcare). Fractions of parasite cell lysate of 500 μl were recovered automatically. The proteins contained in the fractions were precipitated with 10% (w/v) trichloro- acetic acid (TCA) on ice for 30 min and centrifuged. The protein pellet was subjected to Western analysis using anti- PfHsp90, anti-PfHsp70, and anti-PfHop antibodies. The secondary antibody used was HRP-conjugated goat anti- rabbit IgG (Dako, Germany, 1:1:2,000). Immunoprecipitation analysis Parasites released by saponin lysis were resuspended in IP- lysis buffer (150 mM NaCl, 50 mM Tris–HCl, 0.1% NP-40, 1 mM PMSF, and protease inhibitor cocktail (Pierce)), and subjected to three freeze/thaw cycles. A soluble supernatant was collected by centrifugation (4°C, 36,000 ×g). Anti- PfHsp70 antisera were added and the sample split into two aliquots. One aliquot was adjusted to 5 mM ATP and the other received no extra ATP. Antibody binding was allowed to occur overnight at 4°C on a rocking platform. Following this, protein A/G beads (Pierce) were added, and binding was allowed to proceed for 1 h. The immunoprecipitate was then collected by centrifugation at 1,000×g for 5 min at 4°C and the pellet washed extensively with PBS (pH 7.4) before preparation of the samples for SDS-PAGE and Western analysis. Specificity of binding was controlled by monitoring binding to control agarose beads. Results Mapping of the TPR domains in PfHop Based on the Clustal W alignments conducted, PfHop possesses three TPR domains: an N-terminal TPR1 domain composed of residues 7–121, a centrally located TPR2A, at position 243–365 and a C-terminal TPR2B, at position 378–494 (Fig. 1). Residues that are known to be important in the interaction of the TPR domains of mouse Hop with G.W. Gitau et al. either Hsp70 or Hsp90 (Odunuga et al. 2003) are also conserved in PfHop (Fig. 1). Thus, based on sequence alignment data, PfHop presents itself as a typical member of these TPR-rich proteins. Although, Hop proteins whose sequences were analysed appear generally conserved across species, it is interesting to note that there is relative variation between plasmodial Hop proteins in comparison to the human, yeast and mouse homologues even within important functional domains such as the TPR segments and DP motifs (Fig. 1; Carrigan et al. 2005). DP motifs are known to be important in the global fold of Hop and their disruption has been found to impair Hop function (Nelson et al. 2003). The concave surfaces that are formed by the TPR domains of Hop provide interactive sites that can accom- modate specific peptide binding (Scheufler et al. 2000). Based on the three-dimensional model of TPR1 and TPR2A domains, PfHop possesses conserved residues in TPR1 (K11, N15) and TPR2A (K247, N251, K319) that are implicated in the interaction of Hop with Hsp70 and Hsp90 chaperones, respectively (Fig. 2; Scheufler et al. 2000; Odunuga et al. 2004). These residues appear to be surface- exposed on the TPR helical domains, suggesting that they may play an important role in binding PfHsp70 and PfHsp90 chaperones. Overexpression and purification of recombinant PfHop Recombinant PfHop protein was expressed in E. coli XL1 Blue cells as a species of approximately 66 kDa (Fig. 3). However, we also noticed a protein species of approximately 55 kDa, produced in E. coli together with the full length PfHop recombinant protein. The expressed lower molecular weight species, like the full length PfHop protein was produced upon induction of the cells with IPTG and did not appear in the control cells. Anti-PfHop antibodies were able to recognize both the full length Hop protein and the lower molecular weight species as confirmed by Western blotting (Fig. 3b). We concluded that the lower molecular species was either a truncated version of the full length PfHop protein or a product of an incomplete synthesis. We proceeded to purify PfHop by nickel affinity chromatography and were able to recover the full length PfHop protein (Fig. 3a). Fortunately, the species of lower molecular weight did not co-purify with PfHop protein, suggesting that this species had no polyhistidine tag attached (Fig. 3a, c). Therefore, it is likely that the species of lower molecular weight was an N-terminally truncated version of PfHop. Localisation of PfHop The localisation of PfHop in P. falciparum cells at the trophozoite stage was investigated by immunofluorescence