Molecular Farming in Plants

Ursula Hoja, Friedrich‐Alexander‐University Erlangen‐Nuremberg, Erlangen, Germany
Uwe Sonnewald, Friedrich‐Alexander‐University Erlangen‐Nuremberg, Erlangen, Germany

Published online: July 2012

DOI: 10.1002/9780470015902.a0003365.pub2

The commonly used term ‘molecular farming’ describes the large‐scale production of valuable proteins in transgenic plants, including antibodies, vaccines, other pharmaceuticals and industrial proteins. Compared to traditionally used systems such as microbial cultures, plants offer many advantages with respect to economy, quality and safety. Especially attractive is the possibility to power protein production using natural sunlight and atmospheric carbon dioxide. Furthermore, transient expression systems offer the possibility to rapidly produce personalised pharmaceuticals otherwise impossible. Several production systems including algae, mosses, tissue‐ and cell cultures have been reported, enabling contained protein production. Combining these cell‐based production systems with newly developed photo‐bioreactors promises powerful solutions for cost‐efficient and safe manufacturing of valuable proteins. However, constraints concerning protein yield and public acceptance must be overcome before the plant's full potential can be exploited.

Key Concepts:

  • Plant‐based production of proteins can be done in a wide range of systems including different species, tissues and organelles, for each protein the best combination must be identified.
  • Light‐driven production of bioactive‐proteins offers an eco‐friendly and sustainable alternative to conventional production systems.
  • Transient expression of proteins offers the possibility to cost efficiently produce customised pharmaceutical proteins.

Keywords: molecular farming; recombinant proteins; transgenic plants; biotechnology; plant transformation

Figure 1. Different steps to generate transgenic plants suitable for molecular farming of pharmaceuticals: (a) choice of a target gene and insertion into plant expression vector; (b) transfer of the expression construct into Agrobacterium tumefaciens; (c) plant transformation and selection under sterile conditions; (d) generation of transgenic plants; (e) verification of protein accumulation, for example, by Western blot, ELISA, microscopy; (f) functional proof in animal model, for example, testing antibody formation after vaccination; (g) clinical studies; and (h) further development of the product and of the downstream processing technology as well as scaling‐up. P, promoter; G, gene; T, terminator.
Figure 2. Sorting of plastids and isolation of genetically stable transplastomic plants: (a) chloroplast transformation by particle bombardment; (b) incorporation of the transforming DNA in only a single or a few out of several thousand plastid genome copies within a single chloroplast in a leaf cell (blue chloroplast); (c) Enrichment for plants with uniformly transformed plastid genomes during subsequent cell and organelle divisions under antibiotic selection leading to a transplastomic plant (d). P, particle gun; N, nucleus; green, untransformed chloroplast.
Figure 3. Different transient expression systems: (a) Agro‐infiltration: Agrobacteria harbouring the binary construct with gene of interest are infiltrated into leaves leading to protein expression (light red); (b) virus infection: after localised infection, cell‐to‐cell movement, and systemic spread of the virus the protein is expressed at high levels (red) as a fusion to viral coat protein; (c) Agrobacterium‐mediated transfection of virus replicons: the viral vector with the gene of interest is delivered to the plant via Agrobacterium leading to gene amplification within each cell; after cell‐to‐cell movement performed by the replicon the protein of interest is expressed at very high levels (red). Pro, promoter; GoI, gene of interest; T, terminator; TMV, Tobacco mosaic virus; CP, coat protein; MP, movement protein.
Figure 4. N‐linked glycosylation: (a) Structure of typical N‐linked glycans found in plants or mammals and (b) transfer and processing of plant‐specific N‐linked glycans in the ER and Golgi apparatus of plants.
close
 References
    Barta A, Sommergruber K, Thompson D et al. (1986) The expression of nopaline synthase – human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue. Plant Molecular Biology 6: 347–357.
    Beatson R, Sproviero D, Maher J et al. (2011) Transforming growth factor‐β1 is constitutively secreted by Chinese hamster ovary cells and is functional in human cells. Biotechnology and Bioengineering 108(11): 2759–2764, doi 10.1002/bit.23217.
    Biemelt S, Sonnewald U, Galmbacher P, Willmitzer L and Müller M (2003) Production of human papillomavirus Type 16 virus‐like particles in transgenic plants. Journal of Virology 77(17): 9211–9220, doi 10.1128/JVI.77.17.9211‐9220.2003.
    Bińka A, Orczyk W and Nadolska‐Orczyk A (2012) The Agrobacterium‐mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes. Journal of Applied Genetics, 53(1): 1–8, doi 10.1007/s13353‐011‐0064‐y.
    Bock R (2001) Transgenic plastids in basic research and plant biotechnology. Journal of Molecular Biology 312: 425–438.
    Bock R and Warzecha H (2010) Solar‐powered factories for new vaccines and antibiotics. Trends in Biotechnology 28(5): 246–252, doi 10.1016/j.tibtech.2010.01.006.
    Cerutti H, Johnson AM, Gillham NW and Boynton JE (1997) Epigenetic Silencing of a Foreign Gene in Nuclear Transformants of chlamydomonas. Plant Cell 9: 925–945.
    Cheng L, Li HP, Qu B et al. (2010) Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons. Plant Cell Reports 29: 371–381, doi 10.1007/s00299‐010‐0828‐6.
    Conley AJ, Joensuu JJ, Richman A and Menassa R (2011) Protein‐body‐inducing fusions for high‐level production and purification of recombinant proteins in plants. Plant Biotechnology Journal 9: 419–433, doi 10.1111/j.1467‐7652.2011.00596.x.
    Daniell H, Singh ND, Mason H and Streatfield SJ (2009) Plant‐made vaccine antigens and biopharmaceuticals. Trends in Plant Science 14(12): 669–679, doi 10.1016/j.tplants.2009.09.009.
    Decker EL and Reski R (2004) The moss bioreactor. Current Opinion in Plant Biology 7: 166–170, doi 10.1016/j.pbi.2004.01.002.
    De Cosa B, Moar W, Lee SB, Mille M and Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nature Biotechnology 19: 71–74.
    Dyck MK, Lacroix D, Pothier F and Sirard MA (2003) Making recombinant proteins in animals – different systems, different applications. Trends in Biotechnology 21(9): 394–399, doi 10.1016/S0167‐7799(03)00190‐2.
    Franklin S, Ngo B, Efuet E and Mayfield SP (2002) Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant Journal 30(6): 733–744.
    Gomord V, Fitchette AC, Menu‐Bouaouiche L et al. (2010) Plant‐specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnology Journal 8: 564–587, doi 10.1111/j.1467‐7652.2009.00497.x.
    He Y, Jones HD, Chen S et al. (2010) Agrobacterium‐mediated transformation of durum wheat (Triticum turgidum L var. durum cv Stewart) with improved efficiency. Journal of Experimental Botany 61(6): 1567–1581, doi 10.1093/jxb/erq035.
    Kaulfürst‐Soboll H, Mertens M, Brehler R and Schaewen A (2011) Reduction of cross‐reactive determinants in plant foodstaff: elucidation of clinical relevance and implications for allergy diagnosis. PLoS One, doi 10.1371/journal.pone.0017800.
    Lai H, He J, Engle M, Diamond MS and Chen Q (2012) Robust production of virus‐like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant Biotechnology Journal 10(1): 95–104, doi 10.1111/j.1467‐7652.2011.00649.x.
    Maul JE, Lilly JW, Cui L et al. (2002) The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. Plant Cell 14: 2659–2679, doi 10.1105/tpc.006155.
    Nakashima K, Takasaki H, Mizoi J, Shinozaki K and Yamaguchi‐Shinozaki K (2012) NAC Transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta, 1819(2): 97–103, doi 10.1016/j.bbagrm.2011.10.005.
    Oey M, Lohse M, Kreikemeyer B and Bock R (2009) Exhaustion of the chloroplast protein synthesis by massive expression of a highly stable protein antibiotic. Plant Journal 57: 436–445, doi 10.111/j.1365‐313X.2008.03702.x.
    Orzáez D, Granell A and Bláquez MA (2009) Manufacturing antibodies in the plant cell. Biotechnology Journal 4: 1712–1724, doi 10.1002/biot.200900223.
    Paulus KE, Mahler V, Pabst M et al. (2011) Silencing β1,2 xylosyltransferase in transgenic tomato fruits reveals xylose as constitutive component of IgE binding epitopes. Frontiers in Plant Biotechnology. 2: Aricle 42, doi 10.3389/fpls.2011.00042.
    Penney CA, Thomas DR, Deen SS and Walmsley AM (2011) Plant‐made vaccines in support of the Millenium Developmental Goals. Plant Cell Reports 30(5): 789–798, doi 10.1007/s00299‐010‐0995‐5.
    Rasala BA, Muto M, Lee PA et al. (2010) Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnology Journal 8: 719–733, doi 10.1111/j.1467‐7652.2010.00503.x.
    Richter LJ, Thanavala Y, Arntzen CJ and Mason HS (2000) Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nature Biotechnology 18: 1167–1171.
    Sainsbury F, Cañizares MC and Lomonossoff GP (2010) Cowpea masaic virus: the plant virus‐based biotechnology workhorse. Annual Review of Phytopathology 48: 437–455.
    Schaaf A, Tintelnot S, Baur A et al. (2005) Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells. BMC Biotechnology 5: 30, doi 10.1186/1472‐6750‐5‐30.
    Schaefer D, Zryd JP, Knight CD and Cove DJ (1991) Stable transformation of the moss Physcomitrella patens. Molecular and General Genetics 226: 418–424.
    Shrawat AK and Good AG (2011) Agrobacterium tumefaciens‐mediated genetic transformation of cereals using immature embryos. Methods in Molecular Medicine 710(5): 355–372, doi 10.1007/978‐1‐61737‐988‐8_24.
    Sourrouille C, Marquet‐Blouin E, D'Aoust MA et al. (2008) Down‐regulated expression of plant‐specific glycoepitopes in alfalfa. Plant Biotechnology Journal 6: 702–721, doi 10.1111/j.1467‐7652.2008.00353.x.
    Sunil Kumar GB, Ganapathi TR, Revanthi CJ, Srinivas L and Bapat VA (2005) Expression of hepatitis B surface antigen in transgenic banana plants. Planta 222: 484–493, doi 10.1007/s00425‐005‐1556‐y.
    Tharmalingam T, Sunley K, Spearman M and Butler M (2011) Enhanced production of human recombinant proteins from CHO cells grown to high densities in macroporous microcarriers. Molecular Biotechnology 49: 263–276, doi 10.1007/s12033‐011‐9401‐y.
    Tran M, Zhou B, Pettersson PL, Gonzales MJ and Mayfield SP (2009) Synthsis and assembly of a full‐length human monoclonal antibody in algal chlorolasts. Biotechnology and Bioengineering 104(4): 663–673.
    Twyman RM, Stoger E, Schillberg S, Christou P and Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends in Biotechnology 21(12): 570–578, doi 10.1016/j.tibtech.2003.10.002.
    Voinnet O, Rivas S, Mestre P and 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 Journal 33: 949–956.
    Waheed MT, Thönes N, Müller M et al. (2011) Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a Stepp towards cost‐effective second‐generation vaccines. Transgenic Research 20: 271–282, doi 10.1007/s11248‐010‐9415‐4.
    Wally O and Punja ZK (2010) Genetic engineering for increasing fungal and bacterial disease resistance in crop plants. GM Crops 1(4): 199–206.
    Yusibov V, Streatfield SJ and Kushnir N (2011) Clinical development of plant‐produced recombinant pharmaceuticals. Human Vaccines 7(3): 313–321, doi 10.4161/hv/7.3.14207.
    Zimran A, Brill‐Almon E, Chertkoff R et al. (2011) Pivotal trial with plant cell‐expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease. Blood 118: 5767–5773, doi 10.1182/blood‐2011‐07‐366955.
 Further Reading
    Ahmad P, Ashraf M, Younis M et al. (2012) Role of transgenic plants in agriculture and biopharming. Biotechnology Advances 30(3): 524–540, doi 10.1016/j.biotechadv.2011.09.006.
    Basaran P and Rodríguez‐Cerezo E (2008) Plant molecular farming: opportunities and challenges. Critical Reviews in Biotechnologies 28: 153–172, doi 10.1080/07388550802046624.
    Boothe J, Nykiforuk C, Shen Y et al. (2010) Seed‐based expression systems for plant molecular farming. Plant Biotechnology Journal 8: 588–606, doi 10.1111/j.1467‐7652.2010.00511.x.
    Daniell H, Streatfield SJ and Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science 6: 219–226.
    Decker EL and Reski R (2012) Glycoprotein production in moss bioreactors. Plant Cell Reports, 31(3): 453–460, doi 10.1007/s00299‐011‐1152‐5.
    Fischer R, Stoger E, Schillberg S et al. (2004) Plant‐based production of biopharmaceuticals. Current Opinion in Plant Biology 7: 152–158.
    Larrick JW, Lloyd Y, Naftzger C et al. (2001) Production of secretory IgA antibodies in plants. Biomolecular Engineering 18: 87–94.
    Petruccelli S, Otegui MS, Lareu F et al. (2006) A KDEL‐tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnology Journal 2: 511–527, doi 10.1111/j.1467‐7652.2006.00200.x.
    Streatfield SJ (2007) Approaches to achieve high‐level heterologous protein production in plants. Plant Biotechnology Journal 5: 2–15, doi 10.1111./j.1467‐7652.2006.00216.x.

Related Sub-Topics:

Plant Biotechnology | Plant Genetics and Molecular Biology
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Molecular Farming in Plants
 
How to Cite close
Hoja, Ursula, and Sonnewald, Uwe(Jul 2012) Molecular Farming in Plants. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003365.pub2]