Figure 3.

Overexpression and Purification of GST–FtsH.

E. coli cells were transformed with the plant cDNA encoding FtsH carried in pGEX-3X, and expression of GST–FtsH fusion protein was induced by 1 mM isopropyl β-d-thiogalactopyranoside.

(A) Bacterial proteins corresponding to 0.2 mL of cells at an OD₆₀₀ of 1 were subjected to SDS-PAGE and stained with Coomassie blue.

(B) Bacterial proteins corresponding to 50 μL of cells at an OD₆₀₀ of 1 were analyzed by protein gel blot analysis for FtsH.

In (A) and (B), lanes 1, uninduced E. coli cells; lanes 2, cells induced for expression; lanes 3, pellet after sonication and centrifugation; lanes 4, supernatant after sonication and centrifugation; and lanes 5, eluate after glutathione affinity chromatography. GST–FtsH and free GST are indicated by arrowheads, and small amounts of partially cleaved GST–FtsH (97 and 81 kD) are marked with asterisks.