FIG. 5.

Results of an in vitro nPHB granule-binding assay. nPHB granules were isolated from B. megaterium cells grown at 37°C in LB medium supplemented with 2% sodium acetate for 6 h. The purified His-tagged S142C variant (22 ng/μl) of B. megaterium PhaZ1 or His-tagged PhaZ (20 ng/μl) from B. thuringiensis was incubated with nPHB granules (50 μg/μl) at 30°C for 15 min. The incubation mixture was then separated into the supernatant and the granule-containing pellet by centrifugation. Proteins in the supernatant and pellet were subjected to SDS-10% PAGE and analyzed by Western blotting with anti-His tag antibody as the probe. In, input His-tagged protein; T, total proteins before separation into fractions by centrifugation; S, supernatant; P, pellet.