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A protocol for expression of foreign genes in chloroplasts

Abstract

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 6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.

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Figure 1: Schematic representation of the chloroplast integration and expression cassette.
Figure 2: Schematic representation of cloning process to obtain a plastome-specific targeting vector.
Figure 3: Schematic representation of the cloning process to obtain a chloroplast transformation vector.
Figure 4: Selection of transgenic plants.
Figure 5: Evaluation of transgene integration into the chloroplast genome.
Figure 6: Maternal inheritance of transgenes.
Figure 7: Confirmation of transgene expression in chloroplasts.

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References

  1. Koya, V., Moayeri, M., Leppla, S.H. & Daniell, H. Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect. Immun. 73, 8266–8274 (2005).

    Article  CAS  Google Scholar 

  2. Daniell, H. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20, 581–586 (2002).

    Article  CAS  Google Scholar 

  3. Hagemann, R. The sexual inheritance of plant organelles. In Molecular Biology and Biotechnology of Plant Organelles (eds. Daniell, H. & Chase, C.) 93–113 (Springer Publishers, Dordrecht, The Netherlands, 2004).

    Chapter  Google Scholar 

  4. Daniell, H. Transgene containment by maternal inheritance: effective or elusive? Proc. Natl. Acad. Sci. USA 104, 6879–6880 (2007).

    Article  CAS  Google Scholar 

  5. Ruiz, O.N. & Daniell, H. Engineering cytoplasmic male sterility via the chloroplast genome by expression of {beta}-ketothiolase. Plant Physiol. 138, 1232–1246 (2005).

    Article  CAS  Google Scholar 

  6. Arlen, P.A. et al. Field production and functional evaluation of chloroplast-derived interferon alpha2b. Plant Biotechnol. J 5, 511–525 (2007).

    Article  CAS  Google Scholar 

  7. DeCosa, B., Moar, W., Lee, S.B., Miller, M. & Daniell, H. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 19, 71–74 (2001).

    Article  CAS  Google Scholar 

  8. Grevich, J.J. & Daniell, H. Chloroplast genetic engineering: recent advances and future perspectives. Crit. Rev. Plant Sci. 24, 83–107 (2005).

    Article  CAS  Google Scholar 

  9. Daniell, H., Kumar, S. & Dufourmantel, N. Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends Biotechnol. 23, 238–245 (2005).

    Article  CAS  Google Scholar 

  10. Chilton, M.M. & Que, Q. Targeted integration of T-DNA into the tobacco genome at double-stranded breaks: new insights on the mechanism of T-DNA integration. Plant Physiol. 133, 956–965 (2003).

    Article  CAS  Google Scholar 

  11. Dhingra, A., Portis, A.R. Jr. & Daniell, H. Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. Proc. Natl. Acad. Sci. USA 101, 6315–6320 (2004).

    Article  CAS  Google Scholar 

  12. Lee, S.B. et al. Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol. Breed. 11, 1–13 (2003).

    Article  CAS  Google Scholar 

  13. Quesada-Vargas, T., Ruiz, O.N. & Daniell, H. Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation. Plant Physiol. 138, 1746–1762 (2005).

    Article  CAS  Google Scholar 

  14. Staub, J.M. et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18, 333–338 (2000).

    Article  CAS  Google Scholar 

  15. Leelavathi, S. & Reddy, V.S. Chloroplast expression of his-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol. Breed. 11, 49–58 (2003).

    Article  CAS  Google Scholar 

  16. Ruhlman, T., Ahangari, R., Devine, A., Samsam, M. & Daniell, H. Oral delivery of chloroplast derived cholera toxin B-proinsulin protects against development of insulitis in non-obese diabetic mice. Plant Biotechnol. J. 5, 495–510 (2007).

    Article  CAS  Google Scholar 

  17. Daniell, H., Lee, S.B., Panchal, T. & Wiebe, P.O. Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311, 1001–1009 (2001).

    Article  CAS  Google Scholar 

  18. Chebolu, S. & Daniell, H. Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis. Plant Biotechnol. J. 5, 230–239 (2007).

    Article  CAS  Google Scholar 

  19. Birch-Machin, I., Newell, C.A., Hibberd, J.M. & Gray, J.C. Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability. Plant Biotechnol. J. 2, 261–270 (2004).

    Article  CAS  Google Scholar 

  20. Glenz, K. et al. Production of a recombinant bacterial lipoprotein in higher plant chloroplasts. Nat. Biotechnol. 24, 76–77 (2006).

    Article  CAS  Google Scholar 

  21. Fischer, R. & Emans, N. Molecular farming of pharmaceutical proteins. Transgenic Res. 9, 279–299 (2000).

    Article  CAS  Google Scholar 

  22. Cramer, C.L., Boothe, J.G. & Oishi, K.K. Transgenic plants for therapeutic proteins: linking upstream and downstream strategies. Curr. Top. Microbiol. Immunol. 240, 95–118 (1999).

    CAS  PubMed  Google Scholar 

  23. Sidorov, V.A. et al. Technical advance: stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J. 19, 209–216 (1999).

    Article  CAS  Google Scholar 

  24. Ruf, S., Hermann, M., Berger, I.J., Carrer, H. & Bock, R. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat. Biotechnol. 19, 870–875 (2001).

    Article  CAS  Google Scholar 

  25. Daniell, H., Vivekananda, J., Nielsen, B.L., Ye, G.N. & Tewari, K.K. Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proc. Natl. Acad. Sci. USA 87, 88–92 (1990).

    Article  CAS  Google Scholar 

  26. Kunnimalaiyaan, M. & Nielsen, B.L. Fine mapping of replication origins (ori A and ori B) in Nicotiana tabacum chloroplast DNA. Nucleic Acids Res. 25, 3681–3686 (1997).

    Article  CAS  Google Scholar 

  27. Allison, L.A., Simon, L.D. & Maliga, P. Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants. EMBO J. 15, 2802–2809 (1996).

    Article  CAS  Google Scholar 

  28. Ruiz, O.N., Hussein, H.S., Terry, N. & Daniell, H. Phytoremediation of organomercurial compounds via chloroplast genetic engineering. Plant Physiol. 132, 1344–1352 (2003).

    Article  CAS  Google Scholar 

  29. Magee, A. et al. T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype. Transgenic Res. 13, 325–337 (2004).

    Article  CAS  Google Scholar 

  30. Muhlbauer, S.K. & Koop, H.U. External control of transgene expression in tobacco plastids using the bacterial lac repressor. Plant J. 43, 941–946 (2005).

    Article  Google Scholar 

  31. Daniell, H., Carmona-Sanchez, O. & Burns, B. Chloroplast derived antibodies, biopharmaceuticals and edible vaccines. In Molecular Farming (eds. Schillberg, S. & Wiley, V.C.H.) 113–133 (Verlag publishers, Germany, 2004).

    Google Scholar 

  32. Daniell, H. & McFadden, B.A Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts. Proc. Natl. Acad. Sci. USA 84, 6349–6353 (1987).

    Article  CAS  Google Scholar 

  33. Boynton, J.E. et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 1534–1538 (1988).

    Article  CAS  Google Scholar 

  34. Golds, T., Maliga, P. & Koop, H.U. Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Nat. Biotechnol. 11, 95–97 (1993).

    Article  CAS  Google Scholar 

  35. O'Neill, C., Horvath, G.V., Horvath, E., Dix, P.J. & Medgyesy, P. Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplast is an alternative to biolistic delivery systems. Plant J. 3, 729–738 (1993).

    Article  CAS  Google Scholar 

  36. Svab, Z. & Maliga, P. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. USA 90, 913–917 (1993).

    Article  CAS  Google Scholar 

  37. Kumar, S., Dhingra, A. & Daniell, H. Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance. Plant Physiol. 136, 2843–2854 (2004).

    Article  CAS  Google Scholar 

  38. Brosch, M., Krause, K., Falk, J. & Krupinska, K. Analysis of gene expression in amyloplasts of potato tubers. Planta 227, 91–99 (2007).

    Article  CAS  Google Scholar 

  39. Watson, J., Koya, V., Leppla, S.H. & Daniell, H. Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop. Vaccine 22, 4374–4384 (2004).

    Article  CAS  Google Scholar 

  40. Tregoning, J.S. et al. Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res. 31, 1174–1179 (2003).

    Article  CAS  Google Scholar 

  41. Tregoning, J.S. et al. Protection against tetanus toxin using a plant-based vaccine. Eur. J. Immunol. 35, 1320–1326 (2005).

    Article  CAS  Google Scholar 

  42. Molina, A., Hervás-Stubbs, S., Daniell, H., Mingo-Castel, A.M. & Veramendi, J. High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol. J. 2, 141–153 (2004).

    Article  CAS  Google Scholar 

  43. Molina, A., Veramendi, J. & Hervás-Stubbs, S. Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus. Virology 342, 266–275 (2005).

    Article  CAS  Google Scholar 

  44. Fernández-San Millán, A., Mingo-Castel, A., Miller, M. & Daniell, H. A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol. J. 1, 71–79 (2003).

    Article  Google Scholar 

  45. DeGray, G., Rajasekaran, K., Smith, F., Sanford, J. & Daniell, H. Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127, 852–862 (2001).

    Article  CAS  Google Scholar 

  46. Kamarajugadda, S. & Daniell, H. Chloroplast-derived anthrax and other vaccine antigens: their immunogenic and immunoprotective properties. Expert Rev. Vaccines 5, 839–849 (2006).

    Article  CAS  Google Scholar 

  47. Dufourmantel, N. et al. Generation of fertile transplastomic soybean. Plant Mol. Biol. 55, 479–489 (2004).

    Article  CAS  Google Scholar 

  48. Kanamoto, H. et al. Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res. 15, 205–217 (2006).

    Article  CAS  Google Scholar 

  49. Daniell, H., Chebolu, S., Kumar, S., Singleton, M. & Falconer, R. Chloroplast-derived vaccine antigens and other therapeutic proteins. Vaccine 23, 1779–1783 (2005).

    Article  CAS  Google Scholar 

  50. Daniell, H. Production of biopharmaceuticals and vaccines in plants via the chloroplast genome. Biotechnol. J. 1, 1071–1079 (2006).

    Article  CAS  Google Scholar 

  51. Leelavathi, S., Gupta, N., Maiti, S., Ghosh, A. & Reddy, V.S. Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme. Mol. Breed. 11, 59–67 (2003).

    Article  CAS  Google Scholar 

  52. Viitanen, P.V. et al. Metabolic engineering of the chloroplast genome using the Escherichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis. Plant Physiol. 136, 4048–4060 (2004).

    Article  CAS  Google Scholar 

  53. Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 2001).

  54. Costa, G.L. & Weiner, M.P. Bidirectional and directional cloning of PCR products. In PCR Primer 2nd edn. (eds. Dieffenbach C.W. and Dveksler G.S.) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2003).

    Google Scholar 

  55. Lutz, A.L., Svab, Z. & Maliga, P. Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system. Nat. Protoc. 1, 1–11 (2006).

    Article  Google Scholar 

  56. Daniell, H., Ruiz, O.N. & Dhingra, A. Chloroplast genetic engineering to improve agronomic traits. Methods Mol. Biol. 286, 111–137 (2005).

    CAS  PubMed  Google Scholar 

  57. Nugent, G.D., Coyne, S., Nguyen, T.T., Kavanagh, T.T. & Dix, P.J. Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts. Plant Sci. 170, 135–142 (2006).

    Article  CAS  Google Scholar 

  58. Kumar, S., Dhingra, A. & Daniell, H. Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol. Biol. 56, 203–216 (2004).

    Article  CAS  Google Scholar 

  59. Lelivelt, C. et al. Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol. Biol. 58, 763–774 (2005).

    Article  CAS  Google Scholar 

  60. Hou, B.K. et al. Chloroplast transformation in oilseed rape. Transgenic Res. 12, 111–114 (2003).

    Article  CAS  Google Scholar 

  61. Zubko, M., Zubko, E., Zuilen, K., Meyer, P. & Day, A. Stable transformation of petunia plastids. Transgenic Res. 13, 523–530 (2004).

    Article  CAS  Google Scholar 

  62. Okumura, S. et al. Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts. Transgenic Res. 15, 637–646 (2006).

    Article  CAS  Google Scholar 

  63. Nguyen, T.T., Nugent, G., Cardi, T. & Dix, P.J. Generation of homoplasmic plastid transformants of a commercial cultivar of potato (Solanum tuberosum L.). Plant Sci. 168, 1495–1500 (2005).

    Article  CAS  Google Scholar 

  64. Lee, S.M. et al. Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny. Mol. Cells 21, 401–410 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Daniell, H., Datta, R., Varma, S., Gray, S. & Lee, S.B. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat. Biotechnol. 16, 345–348 (1998).

    Article  CAS  Google Scholar 

  66. McBride, K.E. et al. Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology 13, 362–365 (1995).

    CAS  PubMed  Google Scholar 

  67. Kota, M. et al. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc. Natl. Acad. Sci. USA 96, 1840–1845 (1999).

    Article  CAS  Google Scholar 

  68. Dufourmantel, N. et al. Generation and analysis of soybean plastid transformants expressing Bacillus thuringiensis Cry1Ab protoxin. Plant Mol. Biol. 58, 659–668 (2005).

    Article  CAS  Google Scholar 

  69. Chakrabarti, S.K., Lutz, K.A., Lertwiriyawong, B., Svab, Z. & Maliga, P. Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth. Transgenic Res. 15, 481–488 (2006).

    Article  CAS  Google Scholar 

  70. Iamtham, S. & Day, A. Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat. Biotechnol. 18, 1172–1176 (2000).

    Article  CAS  Google Scholar 

  71. Guda, C., Lee, S-B. & Daniell, H. Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts. Plant Cell Rep. 19, 257–262 (2000).

    Article  CAS  Google Scholar 

  72. Lössl, A., Eibl, C., Harloff, H.J., Jung, C. & Koop, H.U. Polyester synthesis in transplastomic tobacco (Nicotiana tabacum L.): significant contents of polyhydroxybutyrate are associated with growth reduction. Plant Cell Rep. 21, 891–899 (2003).

    PubMed  Google Scholar 

  73. Zhang, X.H., Brotherton, J.E., Widholm, J.M. & Portis, A.R. Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis: return of a gene to its pre-endosymbiotic origin. Plant Physiol. 127, 131–141 (2001).

    Article  CAS  Google Scholar 

  74. Roh, K.H. et al. Accumulation of sweet protein monellin is regulated by the psbA 5′UTR in tobacco chloroplasts. J. Plant Biol. 49, 34–43 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

The results reported in this article were supported in part by grants from United States Department of Agriculture 3611-21000-017-00D and National Institutes of Health R01 GM 63879 to H.D. The authors are grateful to Drs. Philip Arlen and Dolendro Singh for critically reading this article and Dr. Arlen for redrawing Figure 7.

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Correspondence to Henry Daniell.

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Verma, D., Samson, N., Koya, V. et al. A protocol for expression of foreign genes in chloroplasts. Nat Protoc 3, 739–758 (2008). https://doi.org/10.1038/nprot.2007.522

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