Figure 5.

Purification of a native Nup84p complex. (a) TEV protease induced release of affinity-purified ProtA-TEV-Nup85p from IgG-Sepharose beads. Schematic representation of the ProtA-TEV-Nup85p fusion protein consisting of two IgG binding domains (ProtA), a 10 residue long spacer sequence, the 7 residue long TEV cleavage sequence (underlined) with the cleavage site (indicated by an arrow), followed by the Nup85p sequence. After TEV elution from the beads, a first and second eluate (E1 and E2) were obtained. Finally, a low pH eluate (HAc) was collected. All eluates were analyzed by SDS-PAGE and Western blotting using anti-Nup85p and anti-ProtA antibodies. The positions of the ProtA-TEV-Nup85p and the cleaved Nup85p, lacking the ProtA tag are indicated. (b) Gel filtration chromatography of the affinity-purified and TEV-released Nup84p complex. The eluates E1 and E2, which were combined and concentrated to yield the load fraction (L) were analyzed by FPLC Superose 6 column chromatography. 0.6-ml fractions were collected and 25 μl of fractions 1–27, E1 and E2, and the load (L, 5% of the injected material) were analyzed by SDS-PAGE and silverstaining. The six subunits containing Nup84p complex peak in fraction 9, the Nup85p-Seh1p heterodimer in fraction 15. Fractions 9 and 15 were also analyzed by Western blotting using anti-Nup85p, anti-Nup84p, anti-Seh1p, and anti-Sec13p antibodies, respectively. The positions of the components of the Nup84p complex are indicated. Heavy and light IgG chains are evident in fractions 15 and 16, and the TEV protease in fractions 19 and 20. The Superose 6 column was calibrated with several molecular weight marker proteins of 670, 440, 252, and 158 kD (Bio-Rad).