Production of recombinant proteins and purification steps.
The upper part of the figure shows a scheme of the purification steps followed to prepare the three types of hetero-oligomers obtained in this work. The numbers indicate the corresponding lane of the representative stained gels shown below. Panel A shows a representative SDS-PAGE gel used for molecular mass estimation, including two sets of standards as indicated in the Materials and Methods section. Samples (30 µl) of key purification steps to produce wild type (B), mutated (C) and truncated MAT II (D) were prepared under standard reducing conditions for SDS-PAGE electrophoresis. Gel lanes correspond to: (1) refolded α2 (10 µg); (2) Q-Sepharose collected peak (10 µg); (3) concentrated Q-Sepharose peak (25 µg); (4) standards; (5) purified hetero-oligomer (10 µg); and (6) purified wild type (10 µg), mutant (5 µg) or truncated β subunit (10 µg). Panels B–D show only the relevant sections of the stained gels for each type of purification; the positions for the 45 and 31 kDa protein standards are indicated on the side of the gels. Dots indicate places where gel lanes have been cropped for clarity.