Optimization of Recombinant Protein Expression in the Chloroplasts of Green Algae

  • Samuel P. Fletcher
  • Machiko Muto
  • Stephen P. Mayfield
Part of the Advances in Experimental Medicine and Biology book series (volume 616)


Through advances in molecular and genetic techniques, protein expression in the chloroplasts of green algae has been optimized for high-level expression. Recombinant proteins expressed in algae have the potential to provide novel and safe treatment of disease and infection where current, high-cost drugs are the only option, or worse, where therapeutic drugs are not available due to their prohibitively high-cost to manufacture. Optimization of recombinant protein expression in Chlamydomonas reinhardtii chloroplasts has been accomplished by employing chloroplast codon bias and combinatorial examination of promoter and UTR combinations. In addition, as displayed by the expression of an anti-herpes antibody, the C. reinhardtii chloroplast is capable of correctly folding and assembling complex mammalian proteins. These data establish algal chloroplasts as a system for the production of complex human therapeutic proteins in soluble and active form, and at significandy reduced time and cost compared to existing production systems. Production of recombinant proteins in algal chloroplasts may enable further development of safe, efficacious and cost-effective protein therapeutics.


Recombinant Protein Therapeutic Protein Codon Bias Recombinant Protein Expression Algal Chloroplast 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    UBS Investment Research: The Q Series™: The State of Biomanufacturing. 2003:1–52.Google Scholar
  2. 2.
    Dove A. Uncorking the biomanufacturing bottleneck. Nat Biotechnol 2002; 20(8):777–779.PubMedCrossRefGoogle Scholar
  3. 3.
    Ma JK, Barros E, Bock R et al. Molecular farming for new drugs and vaccines. Current perspectives on the production of pharmaceuticals in transgenic plants. EMBO Rep 2005; 6(7):593–599.PubMedCrossRefGoogle Scholar
  4. 4.
    Fischer R, Stoger E, Schillberg S et al. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 2004; 7(2):152–158.PubMedCrossRefGoogle Scholar
  5. 5.
    Ko K, Koprowski H. Plant biopharming of monoclonal antibodies. Virus Res 2005; 111(1):93–100.PubMedCrossRefGoogle Scholar
  6. 6.
    Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature 1989; 342(6245):76–78.PubMedCrossRefGoogle Scholar
  7. 7.
    Mason HS, Lam DM, Arntzen CJ. Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA 1992; 89(24):11745–11749.PubMedCrossRefGoogle Scholar
  8. 8.
    Fletcher SP, Geyer BC, Mor TS. Translational control of recombinant human acetylcholinesterase accumulation in plants. submitted to the Journal of Plant PhysiologyGoogle Scholar
  9. 9.
    Staub JM, Garcia B, Graves J et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 2000; 18(3):333–338.PubMedCrossRefGoogle Scholar
  10. 10.
    Borisjuk NV, Borisjuk LG, Logendra S et al. Production of recombinant proteins in plant root exudates. Nat Biotechnol 1999; 17(5):466–469.PubMedCrossRefGoogle Scholar
  11. 11.
    Ellstrand NC. When transgenes wander, should we worry? Plant Physiol 2001; 125(4):1543–1545.PubMedCrossRefGoogle Scholar
  12. 12.
    Quist D, Chapela IH. Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico. Nature 2001; 414(6863):541–543.PubMedCrossRefGoogle Scholar
  13. 13.
    Ma JK, Hikmat BY, Wycoff K et al. Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med 1998; 4(5):601–606.PubMedCrossRefGoogle Scholar
  14. 14.
    Harris E. The Chlamydomonas Sourcebook. In: Academic Press, Inc, 1989:780.Google Scholar
  15. 15.
    Franklin S, Ngo B, Efuet E et al. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J 2002; 30(6):733–744.PubMedCrossRefGoogle Scholar
  16. 16.
    Mayfield SP, Schultz J. Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast. Plant J 2004; 37(3):449–458.PubMedCrossRefGoogle Scholar
  17. 17.
    Mayfield SP, Franklin SE, Lerner RA. Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci USA 2003; 100(2):438–442.PubMedCrossRefGoogle Scholar
  18. 18.
    McBride KE, Svab Z, Schaaf DJ et al. Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology (NY) 1995; 13(4):362–365.PubMedCrossRefGoogle Scholar
  19. 19.
    Ye GN, Hajdukiewicz PT, Broyles D et al. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 2001; 25(3):261–270.PubMedCrossRefGoogle Scholar
  20. 20.
    Lutz KA, Knapp JE, Maliga P. Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol 2001; 125(4):1585–1590.PubMedCrossRefGoogle Scholar
  21. 21.
    Fernandez-San Millan A, Mingo-Castel A, Miller M et al. A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnology Journal 2003; 1(2):71–79.PubMedCrossRefGoogle Scholar
  22. 22.
    Daniell H, Lee SB, Panchal T et al. Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 2001; 311(5):1001–1009.PubMedCrossRefGoogle Scholar
  23. 23.
    Ruf S, Hermann M, Berger IJ et al. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 2001; 19(9):870–875.PubMedCrossRefGoogle Scholar
  24. 24.
    Sidorov VA, Kasten D, Pang SZ et al. Technical Advance: Stable chloroplast transformation in potato: Use of green fluorescent protein as a plastid marker. Plant J 1999; 19(2):209–216.PubMedCrossRefGoogle Scholar
  25. 25.
    Boynton JE, Gillham NW, Harris EH et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 1988; 240(4858):1534–1538.PubMedCrossRefGoogle Scholar
  26. 26.
    Newman SM, Boynton JE, Gillham NW et al. Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: Molecular and genetic characterization of integration events. Genetics 1990; 126(4):875–888.PubMedGoogle Scholar
  27. 27.
    Fischer N, Stampacchia O, Redding K et al. Selectable marker recycling in the chloroplast. Mol Gen Genet 1996; 251(3):373–380.PubMedCrossRefGoogle Scholar
  28. 28.
    Goldschmidt-Clermont M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: A selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res 1991; 19(15):4083–4089.PubMedCrossRefGoogle Scholar
  29. 29.
    Friend PJ, Hale G, Chatenoud L et al. Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection. Transplantation 1999; 68(11):1632–1637.PubMedCrossRefGoogle Scholar
  30. 30.
    Simmons LC, Reilly D, Klimowski L et al. Expression of full-length immunoglobulins in Escherichia coli: Rapid and efficient production of aglycosylated antibodies. J Immunol Methods 2002; 263(1–2):133–147.PubMedCrossRefGoogle Scholar
  31. 31.
    Daniell H, Vivekananda J, Nielsen BL et al. Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proc Natl Acad Sci USA 1990; 87(1):88–92.PubMedCrossRefGoogle Scholar
  32. 32.
    Inada H, Seki M, Morikawa H et al. Existence of three regulatory regions each containing a highly conserved motif in the promoter of plastid-encoded RNA polymerase gene (rpoB). Plant J 1997; 11(4):883–890.PubMedCrossRefGoogle Scholar
  33. 33.
    Eberhard S, Drapier D, Wollman FA. Searching limiting steps in the expression of chloroplast-encoded proteins: Relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. Plant J 2002; 31(2):149–160.PubMedCrossRefGoogle Scholar
  34. 34.
    Barnes D, Franklin S, Schultz J et al. Contribution of 5′-and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol Genet Genomics 2005:1–12.Google Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2007

Authors and Affiliations

  • Samuel P. Fletcher
    • 1
  • Machiko Muto
    • 1
  • Stephen P. Mayfield
    • 1
  1. 1.Department of Cell Biology and The Skaggs Institute for Chemical BiologyThe Scripps Research InstituteLa JollaUSA

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