FIG 2.

Rv2744c forms higher-order homo-oligomeric structures in vitro. (A) Rv2744c-His was purified via Ni affinity chromatography. Protein fractions were resolved by SDS-PAGE to confirm purity (M, molecular mass marker; WCL, whole-cell lysate; FT, flowthrough; W1 to W3, washes 1 to 3; E1 to E4, elutions 1 to 4). (B) Purified Rv2744c-His was resolved on a continuous 10 to 50% sucrose gradient by ultracentrifugation for 16 h at 4°C and 100,000 × g. Fractions were removed from the tops of the gradient tubes, and the absorbance at 280 nm (Abs₂₈₀) was quantified via spectrophotometer to determine protein abundance. Protein standards were also separated in parallel and included β-amylase (200 kDa), thyroglobulin (669 kDa), and blue dextran (2,000 kDa). (C) Fractions containing protein were resolved by SDS-PAGE and probed by Western blotting using mouse anti-His antibody in order to confirm the presence of Rv2744c-His. Only selected fractions are shown. (D and E) Purified Rv2744c isolated from fraction 17 of the sucrose gradient was examined via negative-stain electron microscopy at a magnification of ×100,000 (D) or ×300,000 (E). Regular organized structures of small spheroids and tubular structures with similar diameters were observed.