Figure 2
Expression and localization of the PfI2 gene product by P. falciparum . A. Purified His6-PfI2 separated by 15% SDS-PAGE and blotted onto nitrocellulose (Red Ponceau staining, lane 1) and revealed with mAb anti-His (lane 2) showed a single band at ~ 20 kDa, indicating an anomalous electrophoretic migration of PfI2 (expected size 16.7 kDa). The identity of the purified recombinant PfI2 was further confirmed by MALDI-TOF mass spectrometry. B. Immunoprecipitation of native PfI2 with anti-PfI2 polyclonal antibodies from Plasmodium falciparum extracts, followed by an immunoblot analysis with preimmune serum (lane 1) or with anti-PfI2 antisera (lane 2). C. Detection of PfI2 in total proteins extracted from asynchronous cultures of P. falciparum using PfPP1 column. Total protein extracts (10 mg) pre-cleared on Ni-NTA sepharose beads were incubated overnight with His6-PfPP1 affinity Ni-NTA column as described in Methods. The blots were probed with preimmune serum (lane 1), anti-PfI2 (lane 2) or with anti-His mAb antibodies (lane 3). The blots were revealed as described in Methods. D. Immunoblot analysis of pARL2-PfI2-GFP transfected P. falciparum. Protein extracted from wild-type parasites (lane 1) or from transfected parasites (lane 2) were subjected to western-blotting and probed with anti-GFP mAb antibodies. E. Expression and localization of PfI2-GFP throughout the erythrocytic cell cycle of P. falciparum. Parasites were transfected with pARL2-PfI2-GFP construct as described in Methods and live transfectants were analysed by fluorescence microscopy.