Figure 3

Oligomerization of the wild-type/mutant Alr4641 proteins. (A) In silico analysis. The 203 amino-acid long Alr4641 protein contains an AhpC/TSA domain at its N terminal. The amino acid residue number of the conserved VCP motif, the GGVG and YF motif, and the peroxidatic and resolving cysteines are indicated. (B) Purification of Alr4641 by affinity chromatography. Proteins were resolved by SDS-PAGE and visualized by staining with CBB G-250. Lane 1, whole cell protein extract (10 μg) of un-induced E. coli BL-21/pET4641; lane 2, whole cell protein extract (10 μg) of IPTG-induced E. coli BL-21/pET4641; lane3, clarified cell lysate (10 μg); lane 4, molecular mass marker (SDS-7), lane 5, 200 mM imidazole elution (7.5 μg) and lane 6, 500 mM imidazole elution (6.0 μg). (C) Size exclusion chromatography. The column (Superdex 200 10/300 GL) was pre-equilibrated with buffer (20 mM Tris, 50 mM NaCl, pH 7.2) and a 100μl aliquot of protein (200 μg) was injected. The retention volumes obtained with standard proteins were employed to draw a standard curve (depicted in the insert) that was used to determine the mass of Alr4641 (D) Native PAGE. The purified proteins (10 μg each) were resolved on native polyacrylamide gel (10%) and subsequently stained with CBB. Lane 1, native protein marker; lane 2, Alr4641; lane 3, Alr4641C56S and lane 4, Alr4641C178S. (E) SDS-PAGE analysis of purified proteins (each 10 μg) under reducing or non-reducing conditions. (F) Native PAGE of reduced or oxidized Alr4641. The Alr4641 protein (10 μg) was incubated with either H₂O₂ (10 mM) or DTT (5 mM) for 10 min, resolved on native polyacrylamide gels and visualized by staining with CBB. Lane 1, Alr4641 treated with H₂O₂ (10 mM); lane 2, untreated Alr4641 and lane 3, Alr4641 treated with DTT (5 mM).