α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism
Figure 5
Expression of the native T. vaginalis tvactn3 gene and the cloning and expression of recombinant TvACTN3 (TvACTN3r) and its cellular distribution. (a) Principal motifs of the deduced amino acid sequence of the TvACTN3 protein, including Domain I (DI), which contains two calponin-homology domains (CH), followed by four spectrin repeats (SR) in the central rod domain, and three EF-hands (E); one of them is insensitive to calcium (EF) at the C-terminus. (b) Expression of the 6x-His-tagged TvACTN3r protein. Bacteria E. coli were transformed with pProEX-HTb-TvACTN3 plasmid and protein expression was induced by the addition of 1 mM IPTG for 16 h at 16°C. Protein extracts were separated through 7.5% SDS-PAGE and gels were stained with Coomassie Brilliant Blue (CBB), noninduced bacterial extract (lane 1), IPTG-induced bacterial extract (lane 2). Immunodetection of TvACTN3r polypeptide by WB assays using specific rabbit antibodies: preimmune (PI) serum used as a negative control (lane 3), α-TvACTN3r serum (lane 4). (c) Detection of the native TvACTN3 expression by WB with an α-TvACTN3r antibody. CBB-stained T. vaginalis total extract (lane 1), T. vaginalis total extract transferred onto a NC membrane and incubated with the PI serum (lane 2), or with α-TvACTN3r antibody (lane 3). (d) Trichomonad cell fractionation of parasites grown in iron-depleted (0 μM; lanes 1–3), iron-normal (20 μM; lanes 4–6), and iron-rich (250 μM; lanes 7–9) conditions: Total (T), Membrane (M), and Cytoplasmic (C) fractions were analyzed by WB with α-TvACTN3r, α-TvPFO Ar, and α-TvHSP70r polyclonal antibodies. The last two antibodies were used as controls. Arrowheads indicate the size or position of the protein bands detected by antibodies. kDa, molecular weight standards in kilodaltons. A representative result of three independent experiments with similar results.
