Transgenic 
chloroplast technology could provide a viable solution to the production of 
Insulin-like 
Growth Factor I (IGF-I), 
Human Serum Albumin (HSA), or interferons (IFN) because of hyper-expression capabilities, ability to fold and process eukaryotic proteins with disulfide bridges (thereby eliminating the need for expensive post-purification 
processing). Tobacco is an ideal choice because of its large 
biomass, ease of scale-up (million seeds per 
plant), genetic manipulation and impending need to explore alternate uses for this hazardous 
crop. Therefore, all three 
human proteins will be expressed as follows: a) Develop 
recombinant DNA vectors for enhanced expression via tobacco 
chloroplast genomes b) generate transgenic plants c) characterize transgenic expression of proteins or fusion proteins using molecular and biochemical methods d) large scale purification of therapeutic proteins from transgenic tobacco and comparison of current purification / 
processing methods in E. coli or 
yeast e) Characterization and comparison of therapeutic proteins (yield, purity, functionality) produced in 
yeast or E. coli with transgenic tobacco f) 
animal testing and pre-clinical trials for effectiveness of the therapeutic proteins. 
Mass production of affordable vaccines can be achieved by genetically 
engineering plants to produce recombinant proteins that are candidate vaccine antigens. The B subunits of Enteroxigenic E. coli (LTB) and 
cholera toxin of 
Vibrio cholerae (CTB) are examples of such antigens. When the native LTB 
gene was expressed via the tobacco nuclear 
genome, LTB accumulated at levels less than 0.01% of the total soluble leaf 
protein. Production of effective levels of LTB in plants, required extensive codon modification. Amplification of an unmodified CTB coding sequence in chloroplasts, up to 10,000 copies per 
cell, resulted in the accumulation of up to 4.1% of total soluble 
tobacco leaf protein as oligomers (about 410 fold higher expression levels than that of the unmodified LTB 
gene). PCR and 
Southern blot analyses confirmed stable integration of the CTB 
gene into the 
chloroplast genome. 
Western blot analysis showed that chloroplast synthesized CTB assembled into oligomers and was antigenically identical to purified native CTB. Also, GM1,-
ganglioside binding assays confirmed that chloroplast synthesized CTB binds to the 
intestinal membrane receptor of 
cholera toxin, indicating correct folding and 
disulfide bond formation within the chloroplast. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed. The introduced gene was stably inherited in the subsequent generation as confirmed by PCR and 
Southern blot analyses. Incrased production of an efficient transmucosal carrier molecule and 
delivery system, like CTB, in transgenic chloroplasts makes 
plant based oral vaccines and fusion proteins with CTB needing 
oral administration a much more practical approach.