Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copie s per cell), resulted in the accumulation of up to 4.1% of total soluble to bacco leaf protein as functional oligomers (410-fold higher expression leve ls than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accum ulation of CTB in transgenic chloroplasts, due to aggregation of the oligom eric forms in unboiled samples similar to the aggregation observed for puri fied bacterial antigen. PCR and Southern blot analyses confirmed stable int egration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and w ere antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond fo rmation of CTB pentamers within transgenic chloroplasts. In contrast to stu nted nuclear transgenic plants, chloroplast transgenic plants were morpholo gically indistinguishable from untransformed plants, when CTB was constitut ively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Inc reased production of an efficient transmucosal carrier molecule and deliver y system, like CTB, in transgenic chloroplasts makes plant-based oral vacci nes and fusion proteins with CTB needing oral administration commercially f easible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augur s well for development of vaccines in edible parts of transgenic plants. Fu rthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other fo reign multimeric proteins to be properly expressed and assembled in transge nic chloroplasts. (C) 2001 Academic Press.