Figure 2
Basic native-PAGE pattern and gel-filtration profiles for the reconstitution and purification of A 3 B 3 complexes from A- and B-monomers. (A) Basic native-PAGE pattern stained with CBB R-250. Lane 1, gel-filtration purified mutant A3B3 heterohexamers reconstituted with 200 μM AMP-PNP; lane 2, reconstituted mutant A3B3 heterohexamers with DF heterodimer in the presence of 2 mM AMP-PNP; lane 3, reconstituted mutant A3B3 heterohexamers with DF heterodimer without nucleotides; lane 4, purified A(LV476-7AA)3B(L389A)3 heterohexamer after storage at 4 ºC for 20 days; lane 5, wild-type A3B3 with A and B monomers; and lane 6, wild-type A3B3DF. One μg of protein was loaded in each lane. A* indicates addition of AMP-PNP. (B) Gel-filtration profiles for the purification of mutant A3B3 heterohexamers reconstituted from A and B monomers. Dotted line, A(LV476-7AA)3B3; dashed line, A(LV476-7AA)3B(L389A)3; and solid line, wild-type A3B3. Gel-filtration was performed as described in “Materials and methods”. Mixture of total 6.1 mg (mixing ratio of A- and B-subunits were A:B = 65:52 (1:1 molar ratio)) samples in buffer A were loaded in Superose 6 pg gel-filtration column (500 × 16 mm ID) (GE Healthcare) and eluted with the same buffer. Purified A3B3 complex by gel-filtration was examined on basic native-PAGE as lane 1 (as shown in Figure 2A).