FIGURE 2.
Expression of the PfI3 gene product by P. falciparum. A, purified His fusion PfI3 separated by 15% SDS-PAGE and blotted onto nitrocellulose (Red Ponceau staining, lanes 1–3). B, immunoblot analysis of recombinant PfI3 with rat prebleed sera (lane 1), with rat anti-PfI3 antisera (lane 2), and with mAb anti-His (lane 3) showed a single band at ∼20 Da, indicating an anomalous electrophoretic migration of PfI3 (expected size 13 kDa). The identity of the purified recombinant PfI3 has been further confirmed by MALDI-TOF mass spectrometry. C, detection of endogenous PfI3 in total proteins extracted from asynchronous cultures of P. falciparum. Total protein extracts (10 mg) pre-cleared on Ni-NTA-Sepharose beads were incubated overnight with His6-tagged PfPP1 affinity Ni-NTA column. After washings, proteins eluted with SDS-PAGE loading buffer were migrated and blotted to nitrocellulose. The blots were probed with preimmune serum (lane 1), anti-PfI3 (lane 2), or with anti-His mAb antibodies (lane 3). The blots were revealed as described under “Experimental Procedures.”