FIG. 5.

Applications of PST technologies. (a) Cleavage of PrS₂-tagged protein by factor Xa. PrS₂-HR2898 bound to myxospores was treated with factor Xa on the myxospores at room temperature for 30 min. After the reaction, the mixture was centrifuged at 3,000 × g for 5 min and the supernatant was further centrifuged at 9,000 × g for 20 min and analyzed by SDS-17% PAGE. Lanes: 1, PrS₂-HR2898 purified by one-step purification; 2, supernatant; and 3, myxospore fraction after treatment with factor Xa. (b) Results from a pulldown assay using PST technology. The interaction between PrS₂-Δ45 YihI and Der was examined using the PrS₂ tag and myxospores. The soluble fraction of a cell lysate containing Der was mixed with a lysate containing PrS₂-Δ45 YihI (lane 5) or purified PrS₂ (lane 3), and the mixture was incubated at 4°C for 30 min. Myxospores were added, and the mixture was further incubated at 4°C for 30 min, washed with buffer (10 mM Tris-HCl [pH 7.6] and 1 mM CaCl₂) two times, and checked by SDS-17% PAGE. Lanes: 1, whole-cell lysate containing Der; 2, whole-cell lysate containing PrS₂-Δ45 YihI; 3, Der, PrS₂, and myxospores; 4, Der and myxospores; and 5, Der, PrS₂-Δ45 YihI, and myxospores.