Plasmodium falciparum Hsp70–Hsp90 organising protein FENRKKR), corresponding to amino acids 45–59 and 364– 377 of PfHsp90, respectively. Cloning of PfHop, over-expression and purification of PfHop The coding sequence of PfHop (PlasmoDB accession number: PF14_0324) was PCR amplified from 3D7 gDNA using forward primer (5′-TGCATGCATGGTTAACAAAGAA GAAGCTC-3′) with a SphI site (in bold) and reverse primer (5′-TCTGCAGTTATCGTACCTTCAATATTCCAGC-3′) with a PstI site (in bold) and inserted into the SphI/PstI sites of pQE30 (Qiagen). Plasmid integrity was verified by restriction digest and automated DNA sequencing. To facilitate purification of PfHop recombinant protein, the protein was first overexpressed in E. coli XL1 Blue cells. The E. coli XL1 Blue cells were first transformed with pQE30/PfHop plasmid. Subsequently, one isolated recombinant colony from the YT agar plate was inoculated to YT broth containing 100 μg/ml ampicillin (Roth) followed by incubation at 37°C shaking incubator. At OD600 of 0.6, the cells were induced with 1 mM isopropyl- β-D-1-thiogalactopyranoside [IPTG] (Roth). Five hours after induction, the cells were harvested by centrifugation at 5,000 g for 20 min at 4°C. They were then re-suspended in non-reducing lysis buffer (300 mM NaCl, 10 mM imidazole, 10 mM Tris, pH 8.0, 1 mM phenylmethylsul- phonyl fluoride [PMSF], and 1 mM lysozyme). Cell lysis was allowed to proceed for 20 min at room temperature (22°C), after which the lysate was frozen at −80°C, overnight. The cells were then thawed by mild sonication at amplitude setting of 50 for 7 cycles with 15-second pulse and five-second rest after each cycle. Soluble cell extract was derived after clarification by centrifugation at 5,000 g for 30 min at 4°C. The His-tagged PfHop protein that was in the soluble cell extract was allowed to bind to nickel- charged sepharose beads at 4°C for 4 h. The beads were washed using wash buffer (10 mM Tris, pH 7.5, 300 mM NaCl, 50 mM imidazole), and PfHop recombinant protein was then eluted using elution buffer (10 mM Tris, pH 7.5, 300 mM NaCl, 1 M imidazole). The eluted protein was extensively dialysed at 4°C against a storage buffer (10 mM Tris, pH 7.5, 300 mM NaCl, 50 mM imidazole, 0.8 mM DTT and 10% glycerol). The protein was subsequently concentrated with polyethylene glycol. Construction of plasmid expressing PfHop-GFP The PfHop coding sequence was amplified by PCR from gDNA (clone 3D7) using forward primer (5′-GGCTCGAGATGGT TAACAAAGAAGAAGCTCAGAG-3′) with a XhoI site (in bold) and reverse primer (5′-GGCCTAGGTCGTACCTT CAATATTCCAGC-3′) with a AvrII site (in bold). The 193 successful isolation and amplification of the PCR product were confirmed by agarose gel electrophoresis. The PCR product was recovered by gel purified and restricted with XhoI and AvrII. The PfHop encoding segment was inserted into the pARL-2-GFP vector (Przyborski et al. 2005) between XhoI and AvrII restriction sites located in the multiple cloning site. The product was inserted in frame with a downstream GFP coding sequence to generate pARL-2-GFP/PfHop plasmid construct. Diagnostic restriction digestions using XhoI and AvrII were conducted to confirm the integrity of the pARL-2- GFP/PfHop plasmid construct. The plasmid was also sequenced to confirm that the PfHop encoding segment was inserted in frame with the GFP encoding sequence. Parasite culture and preparation of parasite fractions P. falciparum 3D7 cells were cultured under standard conditions (Przyborski et al. 2005; Trager and Jensen, 1976). Sychronised parasites were harvested using Gelafun- din flotation at the trophoite stage. Trophozoite-infected erythrocytes were subsequently washed in PBS (pH 7.4) and lysed using saponin (0.1%). Lysis was allowed to proceed on ice for 6 min with constant gentle mixing to facilitate the lysis. Parasites were collected by centrifugation at 36,000g for 30 min at 4°C followed by an extensive wash step using PBS (pH 7.4). The parasite lysates were subjected to Western analysis to confirm the expression of PfHop. A host cell protein, glycophorin was also probed as a loading control. Images were acquired using X-ray film (Fuji super RX). Immunofluorescence assays Immunofluorescence assays to investigate the localisation of PfHop and PfHop-GFP in parasite-infected erythrocytes were carried out as previously described (Spork et al. 2009). Following fixation of parasite-infected red blood cells, localisation was examined using anti-PfHop primary antibody (purified IgG fraction, 1:100; this study), poly- clonal anti-PfHsp70 primary antibodies (1:1,000; Pesce et al. 2008) or polyclonal rabbit anti-PfHsp90 antibody (purified IgG fraction 1:100; this study). After extensive washing steps, the erythrocytes were thereafter incubated with cy3-conjugated goat anti-rabbit IgG secondary anti- bodies (1:2,000; DAKO Hamburg, Germany) for 2 h at room temperature. The erythrocytes were observed with fluorescence microscope (cell observer) using a 63× oil immersion objective. The images were acquired using Axiovision software package version 4.8.2. Image J software was used for pseudo-colouring and for preparation of merged co-localisation images. Negative control samples of erythrocytes incubated with respective rabbit pre-immune serum and secondary antibody were used to validate the specificity of the localisation signal.