Fig. 1.

Generation of transgenic parasite expressing RFA-tagged (DDD-GFP) PfLPL1 and localisation of PfLPL1-GFP-DDD fusion protein in transgenic parasites. a Schematic representation of PfLPL1 showing the hydrolase domain and putative peptidase domain. Locations of conserved residues of putative lipase/serine-hydrolase active site are also marked. This fixed pattern GXSXG including the active serine residue is conserved throughout all putative serine hydrolases. b Schematic diagram showing the strategy used to incorporate the regulatable fluorescent affinity (RFA) tag at the 3′ end of endogenous locus of pfLPL1 through single cross over homologous recombination. The pGDB vector contains a blasticidin resistance cassette (BSD) for positive selection. The C-terminal fragment of pfLPL1 gene was cloned in frame with GFP-DDD-tag which consists of E. coli dihydrofolate reductase degradation domain (DDD) with GFP and HA sequences. Schematic diagrams of the pfLPL1genomic loci before and after the homologous recombination are shown. Transgenic parasites obtained by selection over blasticidin, followed by on/off drug cycling to promote integration of the plasmid. Locations of PCR-primers are indicated in the schematic diagram. c PCR based analysis of transgenic parasite cultures to show integration of GFP-DDD-tag in the endogenous loci after drug cycling. Primers combinations are indicated for each panel: 969A/1015A (panel 1); 1016A/1015A (panel 2); 1017A/970A (panel 3); lanes 1, 4, and 7 are for transgenic parasite with integrated plasmid; lanes 2, 5, and 8 are for LPL-pGDB plasmid construct; lanes 3, 6, and 9 are for wild-type 3D7 parasites. d Immunoblot analysis using anti-GFP antibodies and trophozoite stage transgenic parasites expressing PfLPL1-GFP-DDD A band of ~ 70 kDa, representing the fusion protein, and another band of ~ 26 kDa, representing GFP, are detected in the transgenic parasites (lane 2), but not in the wild-type parasites (lane 1). Lower panel shows a immunoblot for the same samples which was probed with anti-BiP antibodies; the BiP was detected in both the transgenic parasite lysate as well as wild-type parasite lysate. e Localization of PfLPL1-GFP-DDD fusion protein in transgenic P. falciparum parasite cultures synchronized and visualized at different developmental stages. Fluorescent microscopic images of live transgenic parasites at ring (16–18 hpi), trophozoite (30 hpi), and schizont (42 hpi) stages. The parasite nuclei were stained with DAPI; fluorescence as well as DIC (differential interference contrast) images were obtained by confocal laser scanning microscope. NIS element software (version 4.0) was used to merge images obtained from different fluorescence/DIC images to produce the merged images. In ring stages, the GFP-fluorescence was observed around the nucleus; in early trophozoite stages, small vesicles are present near the parasitophorous vacuole and in the parasite cytosol. In mature stages, late trophozoite stage, and schizonts, GFP-fluorescence was observed in large vesicular structure in close association with the food-vacuole