Identification of a Novel and Unique Transcription Factor in the Intraerythrocytic Stage of Plasmodium falciparum
Figure 2
Preparation of the recombinant candidate proteins using gene overexpression system in P.falciparum.
(A) Detailed descriptions for each candidate protein. (B) Schematic diagram of plasmid constructs used for the gene overexpression in P. falciparum. Genes for candidate protein Nos. 1, 2, 3 and 4 were inserted into the expression site of the pHC1 vector. For protein purification, the FLAG-tag and His-tag were added to the N and C termini, respectively. The restriction endonuclease recognition sequences shown is X, XhoI. (C) Immunopurification of the recombinant proteins from transgenic parasites. Expressed recombinant proteins were immunopurified with the anti-FLAG antibody and subjected to Western blotting with anti-FLAG antibody. The bands corresponding to the recombinant proteins are indicated with white arrowheads. Sizes are indicated in kDa on the left. (D) EMSA with partially purified recombinant proteins. The same labeled probe was used in Figure 1 in each assay. Lane N is probe only. EMSA was performed with 0.1 µg of each fraction of immunopurified recombinant protein. Lane P is the positive control for the assay with 0.6 µg of nuclear extract derived from the parasite synchronized at the trophozoite/schizont stage. Positions of the free probe and the shifted band are indicated on the left.