FIGURE 9.

Production of ToxP inhibits protein translation. Cell‐free in vitro transcription and translation (IVTT) reactions were incubated with pEXP1‐cbu0665 (250 ng) and decreasing amounts of pET28a(+)‐toxP (250 ng – 8 ng) as a DNA template. Control IVTT reactions were conducted with either only pEXP1‐cbu0665 (250 ng) or pET28a(+)‐toxP (250 ng) plasmids or with all three plasmids together (250 ng of pEXP1‐cbu0665, pET28a(+)‐toxP, and pDEST15‐antitoxP) as a DNA template. (a) Production of CBU0665 (42.5 kDa) and ToxP (18 kDa) was detected by immunoblot of cell‐free IVTT lysates probed simultaneously with anti‐XpressT and anti‐V5 antibodies, respectively. (b) Production of AntitoxP (40.1 kDa) was detected by immunoblot of cell‐free lysates probed with anti‐GST antibody. A dose–response affect was seen on CBU0665 production when decreasing amounts of ToxP were present indicating ToxP inhibited translation of CBU0665. Translation inhibition of CBU0665 production by ToxP was alleviated when AntitoxP was present