Characterization of CTB-HC and CTB-C2 expression in tobacco chloroplasts. (A) Detection of heavy chain fusion protein probed with the CTB antibody. CTB standard, 6.25, 12.5, and 25 ng. Lanes 1 to 4 indicate transplastomic lines. Five micrograms total protein of homogenate fraction per lane was loaded. (B) Detection of heavy chain probed with the A2 antibody. CTB, 25 ng. 1-4, transplastomic lines. Five micrograms total protein of homogenate fraction per lane was loaded. (C) Detection of C2 fusion protein probed with the CTB antibody. CTB standard, 5, 10, and 20 ng. Two micrograms total protein of supernatant or homogenate fraction per lane was loaded. (D) Quantitation of CTB-HC and CTB-C2 expression in tobacco chloroplasts. Proteins were extracted from mature leaves at different time points on the same day. TLP, total leaf protein. (E) Ganglioside GM1 ELISA binding assay. CTB standard (0.1 ng), tobacco CTB-HC (5 µg); tobacco CTB-C2 (1 µg); untransformed tobacco WT (5 µg); BSA, bovine serum albumin (5 µg). (F) Blue native gel electrophoresis and western blot analysis to evaluate pentamer assembly. Pentamer sizes: CTB, 57.5 kDa; CTB-C2: 155 kDa; CTB-HC, 490 kDa. Samples loaded: CTB standard, 100 ng; WT, 40 µg; CTB-HC, 40 µg; CTB-C2, 10 µg. A vertical line is inserted between CTB-HC and CTB-C2 lanes to separate these 2 different blots. H, homogenate fraction; S, supernatant fraction; WT, untransformed WT.