Enhanced Production of Therapeutic Proteins in Plants: Novel Expression Strategies

  • Gowtham Iyappan
  • Rebecca Oziohu Omosimua
  • Ramalingam SathishkumarEmail author


Transient gene expression is a rapid and reproducible approach for the production of therapeutics in large scale. Agrobacterium tumefaciens (diarmed tumorigenicity) is employed by introducing recombinant plasmids into plant intracellular spaces in plant cell through syringe or vacuum infiltration that results in ectopic integration into the plant genome. Gene expression usually occurs 3–4 days post-infiltration. Cytoplasmic proteolytic cleavage and posttranscriptional gene silencing (PTGS) are the limiting factors negatively affecting transgene expression. PTGS may be the limiting factor for heterologous protein expression in plant host system. In order to overcome the issues associated with PTGS and other factors that limit protein expression, several strategies are followed. Transient expression studies for high-level expression or modification in viral vectors by incorporating strong promoter or deconstructed virus and viral regulatory elements/co-expression of PTGS suppressor protein are among a few. The advent of modified yield-giving strategies is also regrettably inadequate to meet the expectations; this may be due to cytoplasmic proteolysis of the heterologous protein. Cytoplasmic persistence of transgenic protein may be improved by intracellular compartmentalization. There are many latent shortcomings addressed in this book chapter on strategies to improve overall yield of high-value protein therapeutics in plants.


Transient expression Vacuum infiltration Posttranscriptional gene silencing Gene expression Viral vectors 



The authors thank financial support to Department of Biotechnology, Bharathiar University, under DST- PURSE scheme.


  1. Alkanaimsh S, Karuppanan K, Guerrero A, Tu A, Hashimoto B, Hwang MS et al (2016) Transient expression of tetrameric recombinant human butyrylcholinesterase in Nicotiana benthamiana. Front Plant Sci 7(June):743. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Almaraz-Delgado AL, Flores-Uribe J, Pérez-España VH, Salgado-Manjarrez E, Badillo-Corona JA (2014) Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii. AMB Express 4(1):57. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Artsaenko O, Kettig B, Fiedler U, Conrad U, Düring K (1998) Potato tubers as a biofactory for recombinant antibodies. Mol Breed 4(4):313–319. CrossRefGoogle Scholar
  4. Bao SUN, Guo-qing SUN, Zhi-gang M, Rui Z, San-dui GUO (2015) A novel constitutive promoter and its downstream 5 ′ UTR derived from cotton (Gossypium spp.) drive high-level gene expression in stem and leaf tissues. J Integr Agric 3119(15):1–11. CrossRefGoogle Scholar
  5. Bäumlein H, Boerjan W, Nagy I, Panitz R, Inzé D, Wobus U (1991) Upstream sequences regulating legumin gene expression in heterologous transgenic plants. Mol Gen Genet MGG 225(1):121–128. CrossRefPubMedGoogle Scholar
  6. Ceresoli V, Mainieri D, Del Fabbro M, Weinstein R, Pedrazzini E (2016) A Fusion between domains of the human bone morphogenetic protein-2 and maize 27 kD γ-zein accumulates to high levels in the endoplasmic reticulum without forming protein bodies in transgenic tobacco. Front Plant Sci 7(March):358. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chen Q, Davis KR (2016) The potential of plants as a system for the development and production of human biologics. F1000Research 5, F1000 Faculty Rev-912. CrossRefGoogle Scholar
  8. Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6(5):219. CrossRefPubMedPubMedCentralGoogle Scholar
  9. De Wilde C, De Neve M, De Ryckea R, Bruynsa A (1996) Intact antigen-binding MAK33 antibody and F ab fragment accumulate in intercellular spaces of Arabidopsis thaliana. Plant Sci 114:233–241. CrossRefGoogle Scholar
  10. De Wilde C, Peeters K, Jacobs A, Peck I, Depicker A (2002) Expression of antibodies and Fab fragments in transgenic potato plants: a case study for bulk production in crop plants. Mol Breed 9:271–282CrossRefGoogle Scholar
  11. Dietzschold B, Gore M, Marchadier D, Niu HS, Bunschoten HM, Otvos L et al (1990) Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine. J Virol 64(8):3804–3809. PubMedPubMedCentralGoogle Scholar
  12. Drake PMW, Chargelegue D, Vine ND, Van Dolleweerd CJ, Obregon P, Ma JK (2002) Transgenic plants expressing antibodies: a model for phytoremediation. FASEB J 16:1855–1860CrossRefGoogle Scholar
  13. Dugdale B, Mortimer CL, Kato M, James TA, Harding RM, Dale JL (2013) In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants. Plant Cell 25(7):2429–2443. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Dugdale B, Mortimer CL, Kato M, James TA, Harding RM, Dale JL (2014) Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants. Nat Protoc 9(5):1010–1027. CrossRefPubMedGoogle Scholar
  15. Dutt M, Erpen L, Ananthakrishnan G, Barthe GA, Brlansky RH, Maiti IB, Grosser JW (2016) Comparative expression analysis of five caulimovirus promoters in citrus. Plant Cell Tissue Org Cult (PCTOC). CrossRefGoogle Scholar
  16. Ferreira-Camargo LS, Tran M, Beld J, Burkart MD, Mayfield SP (2015) Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae. AMB Express 5(1):126. CrossRefPubMedGoogle Scholar
  17. Fiedler U, Phillips J, Artsaenko O, Conrad U (1997) Optimization of scFv antibody production in transgenic plants. Immunotechnology 3:205–216CrossRefGoogle Scholar
  18. Gangl D, Zedler JAZ, Włodarczyk A, Jensen PE, Purton S, Robinson C (2015) Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii. Phytochemistry 110:22–28. CrossRefPubMedGoogle Scholar
  19. Garabagi F, Gilbert E, Loos A, Mclean MD, Hall JC (2012) Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts. Plant Biotechnol J 10(9):1118–1128. CrossRefPubMedGoogle Scholar
  20. Gimpel JA, Mayfield SP (2013) Analysis of heterologous regulatory and coding regions in algal chloroplasts. Appl Microbiol Biotechnol 97(10):4499–4510. CrossRefPubMedGoogle Scholar
  21. Gleba Y, Marillonnet S, Klimyuk V (2004) Engineering viral expression vectors for plants: the “full virus” and the “deconstructed virus” strategies. Curr Opin Plant Biol 7(2):182–188. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Gleba Y, Klimyuk V, Marillonnet S (2005) Magnifection – a new platform for expressing recombinant vaccines in plants. Vaccine 23:2042–2048. CrossRefPubMedGoogle Scholar
  23. Gottschamel J, Lössl A, S, Wang Y, Skaugen M, Bock R, Clarke JL (2016) Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems. Plant Mol Biol:497–512. CrossRefGoogle Scholar
  24. Haikonen T, Rajamäki ML, Valkonen JPT (2013) Improved silencing suppression and enhanced heterologous protein expression are achieved using an engineered viral helper component proteinase. J Virol Methods 193(2):687–692. CrossRefPubMedGoogle Scholar
  25. Hefferon KL (2012a) Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins. Virology 433(1):1–6. CrossRefPubMedGoogle Scholar
  26. Hefferon KL (2012b) Recent advances in virus expression vector strategies for vaccine production in plants. Virol Mycol 01(S1):1–5. CrossRefGoogle Scholar
  27. Hefferon K (2014) Plant virus expression vector development: new perspectives. Biomed Res Int 2014:785382. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Jia H, Van Loock B, Liao M, Verbelen JP, Vissenberg K (2007) Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis. Plant Biotechnol J 5(4):477–482. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53. CrossRefPubMedGoogle Scholar
  30. Kasschau KD, Xie Z, Allen E, Llave C, Chapman EJ, Krizan KA, Carrington JC (2003) P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. Dev Cell 4(2):205–217CrossRefGoogle Scholar
  31. Ko K, Koprowski H (2005) Plant biopharming of monoclonal antibodies. Virus Res 111(1):93–100. CrossRefPubMedGoogle Scholar
  32. Kumagai, M. H., et al. (1993) “Rapid, High-Level expression of biologically active α-trichosanthin in transfected plants by an RNA viral vector.” Proceedings of the national academy of sciences of the United States of America, vol. 90, no. 2, pp. 427–430. JSTOR,
  33. Lacombe S, Bangratz M, Brizard J-P, Petitdidier E, Pagniez J, Sérémé D, Lemesre J-L, Brugidou C (2018) Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate. J Biosci Bioeng 125(1):116–123CrossRefGoogle Scholar
  34. Liu L, Lomonossoff GP (2002) Agroinfection as a rapid method for propagating Cowpea mosaic virus-based constructs. J Virol Methods 105(2):343–348. CrossRefPubMedGoogle Scholar
  35. Lombardi R, Circelli P, Villani ME, Buriani G, Nardi L, Coppola V et al (2009) Mottled crinckle virus, 1–11. CrossRefGoogle Scholar
  36. Lou X, Yao Q, Zhang Z, Peng R, Xiong A, Wang H (2007) Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants. Clin Vaccine Immunol 14(4):464–469. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T et al (2007) Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol 88(5):1460–1469. CrossRefPubMedGoogle Scholar
  38. Mallory AC, Reinhart BJ, Bartel D, Vance VB, Bowman LH (2002) A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco. Proc Natl Acad Sci USA 99(23):15228–15233. CrossRefPubMedGoogle Scholar
  39. Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by agrobacterium. Proc Natl Acad Sci U S A 101(18):6852–6857. CrossRefGoogle Scholar
  40. Marlene P, Ram N, Rodr M, Ayala M, Mart A, Linares M et al (2003) Expression and characterization of an anti- (hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen. Biotechnol Appl Biochem 230:223–230Google Scholar
  41. Merlin M, Gecchele E, Arcalis E, Remelli S, Brozzetti A, Pezzotti M, Avesani L (2016) Enhanced GAD65 production in plants using the MagnICON transient expression system: optimization of upstream production and downstream processing. Biotechnol J 11(4):542–553. CrossRefPubMedGoogle Scholar
  42. Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19(19):5194–5201. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Molina A, Hervás-Stubbs S, Daniell H, Mingo-castel AM, Veramendi J (2004) High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol J 2:141–153. CrossRefPubMedGoogle Scholar
  44. Mori M, Fujihara N, Mise K, Furusawa I (2001) Inducible high-level mRNA amplification system by viral replicase in transgenic plants. Plant J 27(1):79–86. CrossRefGoogle Scholar
  45. Munro S, Pelham HR (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48(5):899–907. CrossRefPubMedGoogle Scholar
  46. Murphy DJ (2007) Improving containment strategies in biopharming. Plant Biotechnol J 5:555–569. CrossRefPubMedGoogle Scholar
  47. Ocampo CG, Lareu JF, Marin Viegas VS, Mangano S, Loos A, Steinkellner H, Petruccelli S (2016) Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana. Plant Biotechnol J:1–11. CrossRefGoogle Scholar
  48. Parmenter DL, Boothe JG, Van Rooijen GJH, Yeung EC, Moloney MM (1995) Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol Biol 29:1167–1180CrossRefGoogle Scholar
  49. Peremarti A, Twyman RM, Gómez-Galera S, Naqvi S, Farré G, Sabalza M et al (2010) Promoter diversity in multigene transformation. Plant Mol Biol 73(4):363–378. CrossRefPubMedGoogle Scholar
  50. Ramirez N, Ayala M, Lorenzo D, Palenzuela D, Herrera L, Doreste V et al (2002) Expression of a single-chain Fv antibody fragment specific for the hepatitis B surface antigen in transgenic tobacco plants. Transgenic Res 11:61–64CrossRefGoogle Scholar
  51. Rasala BA, Muto M, Lee PA, Jager M, Cardoso RMF, Behnke A et al (2011) Production of therapeutic proteins in algea, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J 8(6):719–733. CrossRefGoogle Scholar
  52. Rosales-Mendoza S, Paz-Maldonado LMT, Soria-Guerra RE (2012) Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep 31(3):479–494. CrossRefPubMedGoogle Scholar
  53. Rose AB (2004) The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant J 40(5):744–751. CrossRefPubMedGoogle Scholar
  54. Roth BM, Pruss GJ, Vance VB (2004) Review-Plant viral suppressors of RNA silencing. Virus Res 102(1):97–108. CrossRefPubMedGoogle Scholar
  55. Ruf S, Hermann M, Berger IJ, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19(9):870–875. CrossRefGoogle Scholar
  56. Ruiz C, Pla M, Company N, Riudavets J, Nadal A (2016) High CO2 concentration as an inductor agent to drive production of recombinant phytotoxic antimicrobial peptides in plant biofactories. Plant Mol Biol 90(4–5):329–343. CrossRefPubMedGoogle Scholar
  57. Sainsbury F, Lomonossoff GP (2008) Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 148(3):1212–1218. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Sainsbury F, Thuenemann EC, Lomonossoff GP (2009) PEAQ: Versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7(7):682–693. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Schouten A, Van Engelen FA, De Jong GAMI, Borst-vrenssen AWMT, Zilverentant JF, Bosch D et al (1996) The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Mol Biol 30:781–793CrossRefGoogle Scholar
  60. Schunmann PHD, Coia G, Waterhouse PM (2002) Biopharming the SimpliRED??? HIV diagnostic reagent in barley, potato and tobacco. Mol Breed 9(2):113–121. CrossRefGoogle Scholar
  61. Shah KH, Almaghrabi B, Bohlmann H (2013) Comparison of expression vectors for transient expression of recombinant proteins in plants. Plant Mol Biol Report 31:1529–1538. CrossRefGoogle Scholar
  62. Silhavy D, Molnár A, Lucioli A, Szittya G, Hornyik C, Tavazza M, Burgyán J (2002) A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21(12):3070–3080. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N et al (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18(3):333–338. CrossRefPubMedGoogle Scholar
  64. Torres-Barceló C, Martín S, Daròs JA, Elena SF (2008) From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-pro. Genetics 180(2):1039–1049. CrossRefPubMedPubMedCentralGoogle Scholar
  65. Toth RL, Pogue GP, Chapman S (2002) Improvement of the movement and host range properties of a plant virus vector through DNA shuffling. Plant J 30(5):593–600CrossRefGoogle Scholar
  66. Tran M, Van C, Barrera DJ, Pettersson PL, Peinado CD, Bui J, Mayfield SP (2013) Production of unique immunotoxin cancer therapeutics in algal chloroplasts. Proc Natl Acad Sci USA 110(1):E15–E22. CrossRefPubMedGoogle Scholar
  67. Verch T, Yusibov V, Koprowski H (1998) Expression and assembly of a full-length monoclonal antibody in plants using a plant virus vector. J Immunol Methods 220(1–2):69–75. CrossRefGoogle Scholar
  68. Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33(5):949–956. CrossRefGoogle Scholar
  69. Wannathong T, Waterhouse JC, Young REB, Economou CK, Purton S (2016) New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol:1–11. CrossRefGoogle Scholar
  70. Werner S, Breus O, Symonenko Y, Marillonnet S, Gleba Y (2011) High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector. Proc Natl Acad Sci USA 108(34):14061–14066. CrossRefPubMedGoogle Scholar
  71. Yusibov V, Modelska A, Steplewski K, Agadjanyan M, Weiner D, Hooper DC, Koprowski H (1997) Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc Natl Acad Sci USA 94(11):5784–5788. CrossRefPubMedGoogle Scholar
  72. Zhang X, Mason H (2006) Bean yellow dwarf virus replicons for high-level transgene expression in transgenic plants and cell cultures. Biotechnol Bioeng 93(2):271–279. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  • Gowtham Iyappan
    • 1
  • Rebecca Oziohu Omosimua
    • 2
    • 3
  • Ramalingam Sathishkumar
    • 1
    • 3
    Email author
  1. 1.Plant Molecular Farming Laboratory, DRDO-BU Centre for Life SciencesBharathiar UniversityCoimbatoreIndia
  2. 2.Biotechnology Advanced Research CentreSheda Science and Technology complexFCT-AbujaNigeria
  3. 3.Plant Genetic Engineering Laboratory, Department of BiotechnologyBharathiar UniversityCoimbatoreIndia

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