Protocol

Recombinant Proteins From Plants

Volume 483 of the series Methods in Molecular Biology™ pp 163-192

Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Protocols for Expression, Purification, or Oral Delivery and Functional Evaluation

  • N. Dolendro SinghAffiliated withN. Department of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science
  • , Yi DingAffiliated withDepartment of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science
  • , Henry DaniellAffiliated withDepartment of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science

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Summary

Many vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chloroplast genome and their functionality has been evaluated using in vitro assays in cell cultures (i.e., macrophage lysis assay, inhibition of vesicular stomatitis virus-induced cytopathicity in baby hamster kidney cells, or inhibition of human HIV infection in TZM-BL cells) as well as protection after challenge with bacterial or viral pathogens or antitumor assays or delay the onset of insulitis in suitable animal models. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation technology. Moreover, oral delivery of chloroplast-derived therapeutic proteins eliminates expensive purification steps, cold storage, cold transportation, and delivery via sterile needles, thereby further decreasing their cost. In this chapter, we describe detailed protocols for chloroplast transformation including the construction of chloroplast transformation vectors, delivery of DNA into plant cells using particle bombardment, selection and regeneration of transformants by tissue culture, confirmation of transgene integration into the chloroplast genome and homoplasmy, evaluation of foreign gene expression, purification of foreign protein, or oral delivery via bioencapsulation, functional evaluation using in vitro and in vivo assays, and evaluation of immunity after challenge with pathogens in suitable animal models.

Key words

Chloroplast transformation Transgene containment Genetically modified crops Oral delivery Therapeutic proteins