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Nat Protoc. 2008;3(4):739-58. doi: 10.1038/nprot.2007.522.

A protocol for expression of foreign genes in chloroplasts.

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Department of Molecular Biology and Microbiology, College of Medicine, University of Central Florida, Biomolecular Science, Building #20, Room 336, Orlando, Florida 32816-2364, USA.


Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in approximately 6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.

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