198 Fig. 3 Expression and purification of recombinant PfHop protein. a SDS-PAGE analysis for the expression and purification of recombi- nant PfHop protein. PfHop was expressed in E. coli XL1 Blue cells transformed with pQE30/PfHop plasmid. Lane M represents molec- ular weight markers (in kDa) as indicated on the left hand side; lane C, represents the total extract for cells transformed with pQE30 plasmid after IPTG induction; lane 0, represents total extract for cells transformed with pQE30/PfHop plasmid prior to IPTG induction; lanes 1–ON, represent total extract for cells transformed with pQE30/ PfHop plasmid collected hourly and overnight, respectively; lane F, represents the flow through; lane W, represents wash sample; and lane P, represents eluted PfHop protein. b Western analysis of PfHop using anti-PfHop and c Western analysis of PfHop using anti-His antibodies. The results are representative of at least three independent experiments conserved segments of Hop outside its TPR domains have influence on the overall conformations of the helical turns of the TPR domains, thus are capable of imparting unique structural features to Hop molecules from different species (D'Andrea and Regan 2003). Therefore, the sequence variations observed between PfHop and human Hop may make PfHop amenable for selective inhibition using chemicals that may not interfere with human counterpart. In mouse cells, Hop predominantly occurs in the cytosol under normal growth conditions and shuttles between the G.W. Gitau et al. nucleus and the cytosol (Lässle et al. 1997; Longshaw et al. 2000; 2004). An investigation into the cellular distribution of PfHop in parasites at the trophozoite stage showed that the protein is localised to the cytosol (Fig. 4). As PfHop is implicated in mediating the interaction between PfHsp70 and PfHsp90, we investigated the distribution and co- localisation of these three proteins. Indeed, PfHop dis- played a similar cytosolic localisation profile to PfHsp90 (Fig. 5b), suggesting that the two proteins may associate. Although, PfHop-GFP and PfHsp70 exhibited overlapping cytosolic co-localisation signals, the PfHsp90–PfHop-GFP co-localisation signal was more uniform than that for PfHsp70–PfHop-GFP (Fig. 4b, c). It is possible that PfHop associates more closely with PfHsp90 than with PfHsp70. Cytosolic co-localisation of Hop and Hsp70 homologues from Trypanosoma cruzi, another member of the apico- complexan family, has been reported (Schmidt et al. 2011). Previously, it was proposed that PfHsp70 and PfHsp90 were found to co-elute complexed to a 60 kDa species, which was predicted to be PfHop (Banumathy et al. 2003). Findings from this study point to the possible existence of a PfHop-mediated partnership between PfHsp70 and PfHsp90 as has been previously proposed (Pesce et al. 2010; Shonhai 2010). We performed size exclusion chromatography to inves- tigate if PfHop may occur in complexes with PfHsp90 and PfHsp70 (Fig. 5a). We obtained fractions in which PfHop was present along with PfHsp70 and PfHsp90. This suggests the possible existence of a PfHsp70–PfHop– PfHsp90 complex in P. falciparum. However, we cannot formally rule out the possibility that the three proteins may have occurred in independent complexes that may have given rise to oligomeric units of similar apparent sizes. Unusually, we also obtained a fraction containing a complex of about 440 kDa in which PfHsp90 and PfHsp70 occurred in the absence of PfHop (Fig. 5a). Whilst this may represent possible elution of PfHsp90 and PfHsp70 in independent complexes, the interaction of Hsp70 and Hsp90 through a Hop independent partnership has been reported in other living organisms such as Neurospora (Freitag et al. 1997). Interestingly, we also yielded several fractions of varying sizes in which PfHsp70 and PfHop occurred in the absence of PfHsp90. This was unexpected, as it is known that Hsp90 improves the affinity of Hsp70 for Hop binding (Hernández et al. 2002a, b). However, it is also known that binding of Hsp90 to Hsp70–Hop complex, reduces the number of Hsp70 binding sites on Hop from two to one binding site, although overall the binding of Hsp90 improves affinity of Hsp70 for Hop (Hernández et al. 2002b). Thus, Hsp90 binding may also be perceived as putting a strain on the Hsp70–Hop complex. In addition, in the absence of Hsp90, the Hsp70–Hop complex is stabilised by other protein interactors, particularly Hsp40. This may