Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum

Puran S. Sijwali; Philip J. Rosenthal

doi:10.1073/pnas.0307720101

Fig. 2.

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Verification of falcipain-2 gene disruption. (A) Ethidium bromide-stained agarose gel showing PCR products from WT (lanes 2 and 5) and FP2KO (lanes 3 and 6) gDNAs and pDC-PFP2/GFP^A plasmid DNA (lanes 4 and 7) when primers specific for WT falcipain-2 (1F/2R) and the integration locus (1F/3R) are used. The sizes of DNA markers (lane 1) are indicated (kb). (B) Southern blot of PacI (P) and PacI–FokI (P-F) gDNA digests of WT and FP2KO parasites probed with the falcipain-2 proregion. Fragments of 11.5 kb (P) and 1.6 and 3.9 kb (P-F) for FP2KO gDNA and of 4 kb (P) and 1.9 kb (P-F) for WT gDNA indicate integration of a single copy of pDC-PFP2/GFP^A into the proregion of falcipain-2 by single-crossover homologous recombination. The weaker signals of 15 kb (P) and 4 kb (P-F) correspond to falcipain-2′.(C) Immunoblots of early (24–28 h) trophozoite lysates (TL) and vacuolar fractions (V) of WT and FP2KO parasites (2 × 10⁷ per lane) probed with antibodies to falcipain-2 (FP2) and falcipain-3 (FP3). The positions of molecular mass standards are indicated (kDa).