Figure 2

Expression of PyCPW-WPC-1 mRNA and protein. (A) mRNA expression profiles of pycpw-wpc-1 and other CPW-WPC family members (py00599, py03515, and py04297). RT-PCR was performed using several time points for ookinete culture samples (0, 1, 4, 16, and 24 h). Template amounts were normalized between samples to the expression of pyhsp70, a housekeeping gene. (B) Quantitative analysis of pycpw-wpc family transcription. The bands intensities from Figure 2A were measured and plotted. (C) Recombinant PyCPW-WPC-1 (rPyCPW-WPC-1) synthesis using a wheat germ cell-free protein expression system. From the total lysate, GST-fused rPyCPW-WPC-1 was purified using a glutathione Sepharose column. Then, rPyCPW-WPC-1 was eluted from the column after cleavage by a tobacco etch virus protease. rPyCPW-WPC-1 was detected by SDS-PAGE at an approximate MW of 60 kDa (indicated by arrow). (D) PyCPW-WPC-1 protein expression profiles for all invasive stages. Expression of PyCPW-WPC-1 was examined in parasites at various invasive stages (schizont-enriched sample, sch; ookinete-enriched sample, ook; and salivary gland sporozoites, spz) by western blotting using anti-rPyCPW-WPC-1 antibodies. (E) PyCPW-WPC-1 protein expression during ookinete development. Specific bands corresponding to PyCPW-WPC-1 (indicated by arrow) were observed to be the strongest in 1- and 4-h ookinete culture samples, during which post-transcriptional regulation occurred (also see Figure 2A).