The PrP-like mouse ZIP5 ectodomain purified from mammalian cells gives rise to a homodimeric preparation.

<p>(<b>A</b>) Schematic depiction of the boundaries and molecular organization of the Protein A (PA)-Mm_ZIP5 (residues 20–207) and PA-Tr_PrP-1 (residues 301–424) expression constructs. Both constructs possess an N-terminal Protein A tag followed by a 15 amino acid linker sequence and a TEV protease recognition site. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072446#pone-0072446-g001" target="_blank"><b>Figure 1A</b></a> for color codes indicating domains or secondary structural elements. (<b>B</b>) Strategy employed for the purification of Mm_ZIP5 or Tr_PrP-1 secreted from mammalian cells. (<b>C</b>) SDS-PAGE analysis in the course of purification of Mm_ZIP5 and Tr_PrP-1. Of note, deglycosylated Mm_ZIP5 and Tr_PrP-1 migrate with apparent molecular weights of approximately 20 kDa and 15 kDa, respectively. (<b>D</b>) Far-UV CD spectra of purified Mm_ZIP5 (open circles) and Tr_PrP-1 (open triangles). CD spectra of urea-denatured Mm_ZIP5 (filled circles) and Tr_PrP-1 (filled triangles) are shown for comparison. (<b>E</b>) Urea unfolding titrations of Mm_ZIP5 (open circles) and Tr_PrP-1 (open triangles). Urea denaturation curves (0–9 M urea) were recorded by monitoring the change in the CD signal at 220 nm. (<b>F</b>) Sedimentation equilibrium analysis of ZIP5. The lower panel depicts the sedimentation equilibrium profile of 1 mg/mL ZIP5 plotted as A_{280 nm}<i>vs</i>. radius measured from the center of the rotor. The data were fitted (solid line) to an ideal single component model with a deduced average molecular mass of 40 kDa. The upper panel shows the distribution of the residuals, i.e., the deviation of each measured data point from the best fit.