FIG 1.

Purification and activity analyses of Ct_Tsp. (A) Multiple sequence alignment of the active site in Ct_Tsp and Ec_Prc denoting the conserved catalytic residues S455A (mutated in this study), K481A (mutated in this study), and Q485. (B) 6×His-tagged recombinant proteins were assessed for purity and stability on SDS-PAGE stained with Coomassie brilliant blue for total protein (top left) or probed via Western blotting for the 6×His-tag or a C-terminal region of tail-specific protease (Tsp). (C and D) To assess enzyme activity, 1 μg of enzyme and 3 μg of (C) chlamydial SsrA-tagged green fluorescent protein (GFP) (abbreviated as VAA) or (D) casein were incubated at 37°C for 3 h and the products were resolved on SDS-PAGE and Coomassie-stained. Images represent results from replicate experiments with two independently purified protein preparations. Expected sizes: Ec_Prc, 75.3 kDa after signal processing; and Ct_Tsp, 74.6 kDa. (E) Transcripts of euo, clpP2, omcB, and tsp throughout the Ctr L2 48-h developmental cycle.