Figure 4

Purification of the refolded MB109 functional dimer. (A) Upper panel is a non-reduced SDS-PAGE image showing the aggregation property of the refolded MB109 proteins at pH 5.2, 4.2, 3.2 and 2.2. Black arrow indicates the functional dimer. S: supernatant; P: pellet. Bottom panel is the corresponding densitometry of the functional dimer band in each lane. Black and gray bars represent the relative amounts of functional dimer in supernatants and pellet, respectively. (B) Upper panel, size exclusion chromatographic profile of Superdex 75 and 200 16/60 (connected in series) loaded with acidic fractionated supernatant. Gray bar highlights the fractions pooled for next purification step. Bottom panel is non-reduced SDS-PAGE gel image of each corresponding elution fractions. (C) Ion exchange chromatographic profile of HiTrap SP-FF (1 mL) loaded with the size-exclusion purified sample. Solid line, absorption of 280 nm; dashed line, conductance. Gray bar highlights the fractions containing the functional dimer. (D) Purity analysis of the purified functional dimer on reduced and non-reduced SDS-PAGE gel. The protein was loaded at 0.5, 1, 2, and 10 μg in the absence (left) or presence (right) of 100 mM DTT.