Abstract
The first recombinant proteins were produced in microbes and animal cells cultivated in bioreactors. These systems have become the standard for industrial-scale recombinant protein manufacturing. Later, the production of recombinant proteins was demonstrated in whole plants, which differ morphologically from cell-based systems and require completely different cultivation conditions. Over time, additional plant-based production platforms were established, including hairy roots and cell suspension cultures, which are more similar to conventional cell-based systems in terms of morphology, procedures, and equipment requirements. In this brief overview of the field, we explain why plant-based systems are becoming increasingly attractive for the production of valuable proteins with scientific and commercial applications, but also highlight the challenges that these systems must overcome to achieve more widespread industrial utilization. We discuss various laboratory protocols and approaches for the production of recombinant proteins in plants, as well as strategies to optimize yields, and the regulatory and legal framework.
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References
Herrera-Estrella L, Depicker A, Van Montagu M, Schell J (1983) Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209–213
De Block M, Herrera-Estrella L, van MCE M, Schell J, Zambryski PC (1984) Expression of foreign genes in regenerated plants and in their progeny. EMBO J 3:241681–241689
Stieger M (1987) Versuche zur Integration und Expression chimärer Immunoglobuline in Pflanzen. Dissertation University of Cologne
Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76–78. https://doi.org/10.1038/342076a0
Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A (1990) Production of correctly processed human serum albumin in transgenic plants. Biotechnology 8:217–221. https://doi.org/10.1038/nbt0390-217
Spiegel H, Stöger E, Twyman RM, Buyel JF (2018) Current status and perspectives of the molecular farming landscape. In: Kermode AR, Jiang L (eds) Molecular farming: applications, challenges, and emerging areas. John Wiley & Sons, Inc, Hoboken, New Jersey. https://doi.org/10.1002/9781118801512
Schillberg S, Raven N, Spiegel S, Rasche S, Buntru M (2019) Critical analysis of the commercial potential of plants for the production of recombinant proteins. Front Plant Sci 10:720. https://doi.org/10.3389/fpls.2019.00720
Schillberg S, Finnern R (2021) Plant molecular farming for the production of valuable proteins – critical evaluation of achievements and future challenges. J Plant Physiol 258-259:153359. https://doi.org/10.1016/j.jplph.2020.153359
Spiegel H, Schillberg S, Nölke G (2022) Production of recombinant proteins by agrobacterium-mediated transient expression. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_6
Peyret H, Lomonossoff GP (2022) Specific packaging of custom RNA molecules into cowpea mosaic virus-like particles. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_7
Dickmeis C, Commandeur U (2022) Advanced fusion strategies for the production of functionalized potato virus X virions. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_13
Spiegel H, Boes A, Voepel N, Beiss V, Edgue G, Rademacher R, Sack M, Schillberg S, Reimann A, Fischer R (2015) Application of a scalable plant transient gene expression platform for malaria vaccine development. Front Plant Sci 6:1169. https://doi.org/10.3389/fpls.2015.01169
Capell T, Twyman RM, Armario-Najera V, Ma KCM, Schillberg S, Christou P (2020) Potential applications of plant biotechnology against SARS-CoV-2. Trends Plant Sci 25:635–643. https://doi.org/10.1016/j.tplants.2020.04.009
Rup B, Alon S, Amit-Cohen BC, Brill Almon E, Chertkoff R, Tekoah Y (2017) Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems -the taliglucerase alfa story. PLoS One 12:e0186211. https://doi.org/10.1371/journal.pone.0186211
Szeto TH, Drake PMW, Teh AYH, Falci Finardi N, Clegg AG, Paul MJ, Reljic R, Ma JKC (2022) Production of recombinant proteins in transgenic tobacco plants. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_2
Kapusi E, Stoger E (2022) Molecular farming in seed crops: gene transfer into barley (Hordeum vulgare) and wheat (Triticum aestivum). In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_3
Arcalís E, Pedrazzini E, Hörmann-Dietrich U, Vitale A, Stoger E (2022) Cell biology methods to study recombinant proteins in seeds. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_4
Navarre C, Chaumont F (2022) Production of recombinant glycoproteins in Nicotiana tabacum BY-2 suspension cells. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_5
Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A (2013) Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des 19:5531–5542. https://doi.org/10.2174/1381612811319310008
Buntru M, Vogel S, Finnern R, Schillberg S (2022) Plant-based cell-free transcription and translation of recombinant proteins. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_8
González B, Vazquez-Vilar M, Sánchez-Vicente J, Orzáez D (2022) Optimization of vectors and targeting strategies including GoldenBraid and genome editing tools: GoldenBraid assembly of multiplex CRISPR/Cas12a guide RNAs for gene editing in Nicotiana benthamiana. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_12
Jutras PV, Dodds I, van der Hoorn RAL (2022) A bioluminescent Agrobacterium tumefaciens for imaging bacterial metabolic activity in planta. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_15
Jansing J, Bortesi L (2022) Knockout of glycosyltransferases in Nicotiana benthamiana by genome editing to improve glycosylation of plant-produced proteins. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_14
Buyel JF (2022) Strategies for efficient and sustainable protein extraction and purification from plant tissues. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_9
Spiegel H (2022) Improving recombinant protein recovery from plant tissue using heat precipitation. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_10
Buyel JF (2022) Statistical designs to improve downstream processing. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_16
McNulty MJ, Nandi S, McDonald KA (2022) Technoeconomic modeling and simulation for plant-based manufacturing of recombinant proteins. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_11
Hundleby PAC, D’Aoust MA, Finkle C, Atkins J, Twyman RM (2022) Regulation of molecular farming products. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_17
Thangaraj H (2022) Freedom to operate analysis of molecular farming projects. In: Schillberg S, Spiegel H (eds) Recombinant protein production in plants: Methods and Protocols, Methods in Molecular Biology, vol. 2480. Springer, New York. https://doi.org/10.1007/978-1-0716-2241-4_18
Houdelet M, Galinski A, Holland T, Wenzel K, Schillberg S, Buyel J (2017) Animal component-free agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana. Biotechnol J 12:1600721. https://doi.org/10.1002/biot.201600721
Ullisch D, Müller CA, Maibaum S, Kirchhoff J, Schiermeyer A, Schillberg S, Roberts JL, Treffenfeldt W, Büchs J (2012) Comprehensive characterization of two different Nicotiana tabacum cell lines leads to doubled GFP and HA protein production by media optimization. J Biosci Bioeng 113:242–248. https://doi.org/10.1016/j.jbiosc.2011.09.022
Acknowledgments
We thank Dr. Richard M Twyman for editorial assistance. The authors would like to thank the members of the Newcotiana (760331) and Pharma-Factory (774078) consortia, both funded by the EU, for stimulating discussions on the potential and challenges of plants for the production of recombinant proteins.
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Schillberg, S., Spiegel, H. (2022). Recombinant Protein Production in Plants: A Brief Overview of Strengths and Challenges. In: Schillberg, S., Spiegel, H. (eds) Recombinant Proteins in Plants. Methods in Molecular Biology, vol 2480. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2241-4_1
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DOI: https://doi.org/10.1007/978-1-0716-2241-4_1
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