Expression of M2 protein in the SPP system.

(A) E. coli cells harboring pColdII(sp-4) m2 (from residue 2 to 49 of M2 protein) and pACYCmazF were grown at 37°C to OD600 = 0.5∼0.6, followed by cold-shock at 15°C for about 60 min. 1 mM of IPTG was added at 0 hr (Lane 1) for induction of M2 protein and MazF. Expression of M2 protein in the SPP system was examined in the presence of amantadine at different concentrations. Lane 2, 0 μM; Lane 3, 50 μM; Lane 4, 100 μM; Lane 5, 200 μM. After overnight incubation for 19 hours, cells from each culture were collected and subjected to SDS-PAGE. (B) Expression of M2 protein in the presence of other compounds besides amantadine. The final concentration of each compound in the culture is 50 μM. The experiments were carried out as described in (A). Lane 1, 1 mM IPTG is added to the culture at 0 hr, Lane 2: C, control without any additional compounds. Lane 3, compound 10, Lane 4, compound 15, Lane 5, compound 34, Lane 6, compound 35, Lane 7, compound 282, Lane 8, compound 293, Lane 9, compound 314, Lane 10, A, amantadine. (C) Expression of AcGFP-M2 fusion protein in the SPP system was carried out as described in (B). Positions of M2 protein and AcGFP-M2 fusion protein are indicated by arrowheads. (D) Cell density was measured as OD600 of each overnight culture that expressing AcGFP-M2 fusion protein, and plotted as histogram corresponding to the compounds added. (E) Growth curve of cultures to express M2 or AcGFP-M2 fusion protein. Cultures were started at 0 hr and the following experiment procedures are similar to that described in (A). OD 600 of each culture is measured at every time point. M2 protein was induced at 5 hr with (▪) or without (•) amantadine. AcGFP-M2 fusion protein was induced at 5 hr with (♦) or without amantadine (▴).