Chloroplast-derived vaccine antigens and other therapeutic proteins
Introduction
There are many advantages in producing human therapeutic proteins in plants. Plant systems provide opportunity for low cost production, the ability to carry out post-translational modifications and minimize the risk of contamination from potential human pathogens. If the protein is delivered orally, then the expensive step of purifying the recombinant protein could be eliminated. Other advantages of plant derived therapeutic proteins include convenient storage, elimination of hospitals and health professionals for their delivery and the use of renewable resources for their production.
Chloroplast genetic engineering offers several unique advantages over nuclear transformation, which includes very high expression levels of the recombinant proteins. For example, Bacillus thuringiensis (Bt) cry2Aa2 protein was expressed in transgenic chloroplasts up to 46.1% of the total leaf protein [1] or 500–4000-fold more vaccine antigens [2], [3] or 500-fold higher human blood proteins [4] than nuclear transgenic plants. Chloroplast genomes defy the laws of Mendelian inheritance in that they are maternally inherited and thus minimize the out cross of transgenes via pollen [5]. Furthermore, several challenges in nuclear genetic engineering could be eliminated including position effect [2], [6] due to site-specific integration of transgenes by homologous recombination. The problem of gene silencing both at transcriptional and translational levels has not been observed in transgenic chloroplasts in spite of high levels of translation, up to 46.1% tlp [1] or transcription, 169-fold higher rate than nuclear transgenic plants [7].
Also chloroplasts can process eukaryotic proteins, including interferons, vaccine antigens and human somatotropin with correct folding of subunits and formation of disulfide bridges. Chaperones present in chloroplasts facilitate the correct folding and assembly of complex multi-subunit proteins. The major cause for the high cost of pharmaceutical protein production is the purification of the recombinant proteins. Therefore, novel protein purification strategies may be used that does not require the use of expensive column chromatography. For example, the use of a protein-based polymer with inverse temperature transition properties to purify pro-insulin expressed by chloroplast vectors by simple centrifugation is a major accomplishment [8]. Also, oral delivery of the recombinant protein could cut down 90% of the production costs.
Section snippets
Oral delivery of vaccines
One important requirement to achieve oral delivery is the use of antibiotic free selectable markers. The spinach betaine aldehyde dehydrogenase (BADH) gene has been developed as a selectable marker, which facilitates the visual selection of transgenic green cells from non-transgenic yellow cells. The toxic betaine aldehyde (BA) is converted to non-toxic glycine betaine by the chloroplast BADH enzyme. Glycine betaine also serves as osmoprotectant conferring the highest level of salt tolerance,
Cholera toxin B (CTB) antigen
Cholera toxin B sub-unit (CTB) of Vibrio cholerae, a candidate vaccine antigen has been expressed in chloroplasts and this resulted in accumulation of up to 31.1% of total soluble protein as functional oligomers [2], [3]. Also, CTB synthesized from transgenic chloroplasts assembled into functional oligomers and were antigenically identical to purified native CTB. Binding assays have confirmed that chloroplast synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, also
Chloroplast-derived therapeutic proteins
Several examples of human therapeutic proteins expressed in transgenic chloroplasts are listed in Table 1. Expression levels depend on the site of integration, regulatory elements used to enhance transcription/translation and the stability of the foreign protein. Genes coding for 20 amino acids (magainin) or 83 kDa (PA) have been expressed in transgenic chloroplasts. Most of them have been shown to be properly folded and fully functional. The following are a few illustrations from the Daniell
Epilogue
Chloroplast system is most suitable for high-level expression and economical production of therapeutic proteins in an environmentally friendly manner. However, the cost for purification of these proteins can be eliminated if they are orally delivered or minimized by the use of novel purification strategies. Oral delivery of therapeutic proteins is emerging as a new alternative for medical treatment and will benefit those who cannot afford the high cost of current treatments.
Acknowledgements
Results of investigations from the Daniell laboratory are supported in part by the United States Department of Agriculture 3611-21000-017-00D and the National Institutes of Health R01 GM 63879 grants.
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