Recombinant Proteins From Plants pp 163-192

Part of the Methods in Molecular Biology™ book series (MIMB, volume 483)

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

  • N. Dolendro Singh
  • Yi Ding
  • Henry Daniell
Protocol

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 

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • N. Dolendro Singh
    • 1
  • Yi Ding
    • 2
  • Henry Daniell
    • 3
  1. 1.N. Department of Molecular Biology and MicrobiologyUniversity of Central Florida, Biomolecular ScienceOrlandoU.S.A.
  2. 2.Department of Molecular Biology and MicrobiologyUniversity of Central Florida, Biomolecular ScienceFLU.S.A.
  3. 3.Department of Molecular Biology and MicrobiologyUniversity of Central Florida, Biomolecular ScienceOrlandoU.S.A.

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