FIG. 2.

Overproduction and purification of Ppr1p. (A) Time course of Ppr1p expression in baculovirus-infected insect cells. Cultures of High Five insect cells (50% confluent) were infected at time zero with recombinant baculovirus containing the PPR1 gene under the control of the polyhedrin promoter. Samples of the cells (lanes C) or the culture supernatant (lanes S) were taken at the times indicated and analyzed by Western blotting. (B) Purification of Ppr1p. Insect cells infected with recombinant PPR1 baculovirus were harvested 2 days postinfection. Cell extracts were prepared, and soluble protein was applied to a Ni²⁺-NTA agarose column. The column flowthrough is shown in lane 2. The column was washed with loading buffer (lanes 3 and 4) and then with buffer containing 30 mM imidazole (lane 5 to 7). Ppr1p was eluted with buffer containing 250 mM imidazole (lane 8 to 15). Samples of each fraction were run on an SDS-polyacrylamide gel that was stained with Coomassie brilliant blue. Sizes of molecular weight standards (M; in kilodaltons) are indicated. (C) Western blot analysis of the purification of Ppr1p. Samples from lanes 2, 3, 5, and 12 in panel B were separated by SDS-polyacrylamide gel electrophoresis and subjected to Western blotting. Sizes of molecular weight standards are indicated.