Abstract
Hairy roots (HR) are a promising plant tissue-based platform for the production of biopharmaceuticals as the secretion of recombinant proteins in the culture medium may simplify the purification and recovery steps. In this study, Nicotiana benthamiana HR cultures were generated to produce the secretory version of an infectious bursal disease virus chimeric antigen (PD-FcY) of interest for the formulation of poultry vaccines, with the aim of optimizing production and purification. Recombinant protein accumulation kinetics in the medium after induction with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), whose effects were still poorly characterized, was firstly investigated using a HR clone expressing the red fluorescent protein. The results demonstrated that HR cultures induced once and grown 21 days efficiently secrete the protein and, even after replacement of the medium with a hormone-free counterpart, maintain the same secretion rate for several days. This protocol applied to HR expressing PD-FcY resulted in similar accumulation kinetics and allowed to obtain protein recovery concentrations of ~ 4 mg/l. A downstream processing procedure consisting in recombinant product concentration through ultrafiltration followed by two sequential chromatographic steps led to final yields of 0.8 mg purified PD-FcY/l of culture. The HR-derived product showed a similar glycosylation pattern compared to the counterpart obtained from agroinfiltrated N. benthamiana, but higher quality in terms of purity and protein degradation, and was also shown to retain full antigenic potential. In conclusion, the proposed expression and purification strategy holds promises for the development of an innovative platform to produce low-cost subunit vaccines.
Key message
Hairy roots secreting a functional recombinant antigen for veterinary applications were developed and conditions enhancing production and recovery from the culture medium defined, improving the efficacy of this plant-production platform.
Similar content being viewed by others
References
Akita EM, Nakai S (1993) Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain. J Immunol Methods 160:207–214. https://doi.org/10.1016/0022-1759(93)90179-B
Bordelon T, Bobay B, Murphy A, Reese H, Shanahan C, Odeh F, Broussard A, Kormos C, Menegatti S (2019) Translating antibody-binding peptides into peptoid ligands with improved affinity and stability. J Chromatogr A 1602:284–299. https://doi.org/10.1016/j.chroma.2019.05.047
Buyel JF, Twyman RM, Fischer R (2015) Extraction and downstream processing of plant-derived recombinant proteins. Biotechnol Adv 33:902–913. https://doi.org/10.1016/j.biotechadv.2015.04.010
Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882. https://doi.org/10.1073/pnas.082243699
D’Aoust M-A, Couture MM-J, Charland N, Trépanier S, Landry N, Ors F, Vézina L-P (2010) The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza. Plant Biotechnol J 8:607–619. https://doi.org/10.1111/j.1467-7652.2009.00496.x
Desiderio A, Salzano AM, Scaloni A, Massa S, Pimpinella M, De Coste V, Pioli C, Nardi L, Benvenuto E, Villani ME (2019) Effects of simulated space radiations on the tomato root proteome. Front Plant Sci. https://doi.org/10.3389/fpls.2019.01334
Donini M, Lombardi R, Lonoce C, Di Carli M, Marusic C, Morea V, Di Micco P (2015) Antibody proteolysis: a common picture emerging from plants. Bioengineered 6:299–302. https://doi.org/10.1080/21655979.2015.1067740
Donini M, Marusic C (2018) Hairy roots as bioreactors for the production of biopharmaceuticals. In: Hairy roots. Springer, Singapore, pp 213–225
Doran PM (2013) Therapeutically important proteins from in vitro plant tissue culture systems. Curr Med Chem 20:1047–1055. https://doi.org/10.2174/0929867311320080008
Drake PMW, Barbi T, Sexton A, McGowan E, Stadlmann J, Navarre C, Paul MJ, Ma JKC (2009) Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB J 23:3581–3589. https://doi.org/10.1096/fj.09-131771
Du Y, Scheres B (2018) Lateral root formation and the multiple roles of auxin. J Exp Bot 69:155–167. https://doi.org/10.1093/jxb/erx223
Ele Ekouna J-P, Boitel-Conti M, Lerouge P, Bardor M, Guerineau F (2017) Enhanced production of recombinant human gastric lipase in turnip hairy roots. Plant Cell, Tissue Organ Cult 131:601–610. https://doi.org/10.1007/s11240-017-1309-1
Fernández-Arias A, Risco C, Martínez S, Albar JP, Rodríguez JF (1998) Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles. J Gen Virol 79:1047–1054. https://doi.org/10.1099/0022-1317-79-5-1047
Gao Y, Zhao X, Sun C, Zang P, Yang H, Li R, Zhang L (2015) A transgenic ginseng vaccine for bovine viral diarrhea. Virol J 12:1–7. https://doi.org/10.1186/s12985-015-0301-9
Goulet C, Khalf M, Sainsbury F, D’Aoust M-A, Michaud D (2012) A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast. Plant Biotechnol J 10:83–94. https://doi.org/10.1111/j.1467-7652.2011.00643.x
Gurusamy PD, Schäfer H, Ramamoorthy S, Wink M (2017) Biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of Nicotiana tabacum L. PLoS ONE 12:1–23. https://doi.org/10.1371/journal.pone.0182367
Häkkinen ST, Raven N, Henquet M, Laukkanen M-L, Anderlei T, Pitkänen J-P, Twyman RM, Bosch D, Oksman-Caldentey K-M, Schillberg S, Ritala A (2014) Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody. Biotechnol Bioeng 111:336–346. https://doi.org/10.1002/bit.25113
He C-Q, Ma L-Y, Wang D, Li G-R, Ding N-Z (2009) Homologous recombination is apparent in infectious bursal disease virus. Virology 384:51–58. https://doi.org/10.1016/J.VIROL.2008.11.009
Hu Z-B, Du M (2006) Hairy root and its application in plant genetic engineering. J Integr Plant Biol 48:121–127. https://doi.org/10.1111/j.1744-7909.2006.00121.x
Huet Y, Ekouna JPE, Caron A, Mezreb K, Boitel-Conti M, Guerineau F (2014) Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots. Biotechnol Lett 36:181–190. https://doi.org/10.1007/s10529-013-1335-y
Hunter PR, Craddock CP, Di Benedetto S, Roberts LM, Frigerio L (2007) Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in arabidopsis cells. Plant Physiol 145:1371–1382. https://doi.org/10.1104/pp.107.103945
Kim SR, Sim JS, Ajjappala H, Kim YH, Hahn BS (2012) Expression and large-scale production of the biochemically active human tissue-plasminogen activator in hairy roots of Oriental melon (Cucumis melo). J Biosci Bioeng 113:106–111. https://doi.org/10.1016/j.jbiosc.2011.09.008
Kolotilin I, Topp E, Cox E, Devriendt B, Conrad U, Joensuu J, Stöger E, Warzecha H, McAllister T, Potter A, McLean MD, Hall JC, Menassa R (2014) Plant-based solutions for veterinary immunotherapeutics and prophylactics. Vet Res 45:1–12. https://doi.org/10.1186/s13567-014-0117-4
Li C, Wen Y, He Y, Zhu J, Yin X, Yang J, Zhang L, Song L, Xia X, Yu R (2019) Purification and characterization of a novel β-1,3-glucanase from Arca inflata and its immune-enhancing effects. Food Chem 290:1–9. https://doi.org/10.1016/j.foodchem.2019.03.131
Liew PS, Hair-Bejo M (2015) Farming of plant-based veterinary vaccines and their applications for disease prevention in animals. Adv Virol. https://doi.org/10.1155/2015/936940
Liu C, Towler MJ, Medrano G, Cramer CL, Weathers PJ (2009) Production of mouse interleukin-12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift reactor. Biotechnol Bioeng 102:1074–1086. https://doi.org/10.1002/bit.22154
Lombardi R, Villani ME, Di Carli M, Brunetti P, Benvenuto E, Donini M (2010) Optimisation of the purification process of a tumour-targeting antibody produced in N. Benthamiana using vacuum-agroinfiltration. Transgenic Res 19:1083–1097
Lonoce C, Marusic C, Morrocchi E, Salzano AM, Scaloni A, Novelli F, Pioli C, Feeney M, Frigerio L, Donini M (2019) Enhancing the secretion of a glyco-engineered anti-CD20 scFv-Fc antibody in hairy root cultures. Biotechnol J 14:1800081. https://doi.org/10.1002/biot.201800081
Lonoce C, Salem R, Marusic C, Jutras PV, Scaloni A, Salzano AM, Lucretti S, Steinkellner H, Benvenuto E, Donini M (2016) Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures. Biotechnol J 11:1209–1220. https://doi.org/10.1002/biot.201500628
Madeira LM, Szeto TH, Henquet M, Raven N, Runions J, Huddleston J, Garrard I, Drake PMW, Ma JK-C (2016) High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures. Plant Biotechnol J 14:615–624. https://doi.org/10.1111/pbi.12407
Mai NTP, Boitel-Conti M, Guerineau F (2016) Arabidopsis thaliana hairy roots for the production of heterologous proteins. Plant Cell Tissue Organ Cult 127:489–496. https://doi.org/10.1007/s11240-016-1073-7
Pham NB, Schäfer H, Wink M (2012) Production and secretion of recombinant thaumatin in tobacco hairy root cultures. Biotechnol J 7:537–545. https://doi.org/10.1002/biot.201100430
Rage E, Drissi Touzani C, Marusic C, Lico C, Göbel T, Bortolami A, Bonfante F, Salzano AM, Scaloni A, Fellahi S, El Houadfi M, Donini M, Baschieri S (2019) Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins. Appl Microbiol Biotechnol 103:7491–7504. https://doi.org/10.1007/s00253-019-09992-9
Salzano AM, Novi G, Arioli S, Corona S, Mora D, Scaloni A (2013) Mono-dimensional blue native-PAGE and bi-dimensional blue native/urea-PAGE or/SDS-PAGE combined with nLC–ESI-LIT-MS/MS unveil membrane protein heteromeric and homomeric complexes in Streptococcus thermophilus. J Proteomics 94:240–261. https://doi.org/10.1016/J.JPROT.2013.09.007
Saraswat M, Musante L, Ravidá A, Shortt B, Byrne B, Holthofer H (2013) Preparative purification of recombinant proteins: current status and future trends. Biomed Res Int. https://doi.org/10.1155/2013/312709
Schiermeyer A (2020) Optimizing product quality in molecular farming. Curr Opin Biotechnol 61:15–20. https://doi.org/10.1016/j.copbio.2019.08.012
Schillberg S, Raven N, Fischer R, Twyman R, 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
Schillberg S, Raven N, Spiegel H, Rasche S, Buntru M (2019) Critical analysis of the commercial potential of plants for the production of recombinant proteins. Front Plant Sci. https://doi.org/10.3389/fpls.2019.00720
Shams MV, Nazarian-Firouzabadi F, Ismaili A, Shirzadian-Khorramabad R (2019) Production of a recombinant dermaseptin peptide in nicotiana tabacum hairy roots with enhanced antimicrobial activity. Mol Biotechnol 61:241–252. https://doi.org/10.1007/s12033-019-00153-x
Sharp JM, Doran PM (2001a) Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures. Biotechnol Prog 17:979–992. https://doi.org/10.1021/bp010104t
Sharp JM, Doran PM (2001b) Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol Bioeng 73:338–346. https://doi.org/10.1002/bit.1067
Sheng L, He Z, Chen J, Liu Y, Ma M, Cai Z (2017) The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk. Int J Biol Macromol 96:129–136. https://doi.org/10.1016/j.ijbiomac.2016.12.043
Sheshukova EV, Komarova TV, Dorokhov YL (2016) Plant factories for the production of monoclonal antibodies. Biochem 81:1118–1135
Singh A, Srivastava S, Chouksey A, Panwar BS, Verma PC, Roy S, Singh PK, Saxena G, Tuli R (2015) Expression of rabies glycoprotein and ricin toxin B chain (RGP–RTB) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies. Mol Biotechnol 57:359–370. https://doi.org/10.1007/s12033-014-9829-y
Skarjinskaia M, Ruby K, Araujo A, Taylor K, Gopalasamy-Raju V, Musiychuk K, Chichester JA, Palmer GA, de la Rosa P, Mett V, Ugulava N, Streatfield SJ, Yusibov V (2013) Hairy roots as a vaccine production and delivery system. Adv Biochem Eng Biotechnol 134:115–134. https://doi.org/10.1007/10_2013_184
Suzuki N, Lee YC (2004) Site-specific N-glycosylation of chicken serum IgG. Glycobiology 14:275–292. https://doi.org/10.1093/glycob/cwh031
Torres-Martínez HH, Rodríguez-Alonso G, Shishkova S, Dubrovsky JG (2019) Lateral root primordium morphogenesis in angiosperms. Front Plant Sci 10:206. https://doi.org/10.3389/fpls.2019.00206
Van Reis R, Zydney A (2001) Membrane separations in biotechnology. Curr Opin Biotechnol 12:208–211. https://doi.org/10.1016/S0958-1669(00)00201-9
Viertlboeck BC, Göbel TW (2011) The chicken leukocyte receptor cluster. Vet Immunol Immunopathol 144:1–10. https://doi.org/10.1016/j.vetimm.2011.07.001
Villani ME, Morgun B, Brunetti P, Marusic C, Lombardi R, Pisoni I, Bacci C, Desiderio A, Benvenuto E, Donini M (2009) Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies. Plant Biotechnol J 7:59–72. https://doi.org/10.1111/j.1467-7652.2008.00371.x
Wilken LR, Nikolov ZL (2012) Recovery and purification of plant-made recombinant proteins. Biotechnol Adv 30:419–433. https://doi.org/10.1016/j.biotechadv.2011.07.020
Xu J, Dolan MC, Medrano G, Cramer CL, Weathers PJ (2012) Green factory: plants as bioproduction platforms for recombinant proteins. Biotechnol Adv 30:1171–1184. https://doi.org/10.1016/j.biotechadv.2011.08.020
Xu J, Zhang N (2014) On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect. Pharm Bioprocess 2:499–518. https://doi.org/10.4155/pbp.14.32
Zhu J, Arena S, Spinelli S, Liu D, Zhang G, Wei R, Cambillau C, Scaloni A, Wang G, Pelosi P (2017) Reverse chemical ecology: olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles. Proc Natl Acad Sci 114:E9802–E9810. https://doi.org/10.1073/PNAS.1711437114
Acknowledgements
This study was funded by AVIAMED project through the ERANET ARIMNet2 2015 Call by the following funding agency: Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF). ARIMNet2 (ERA-NET) has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127/182.
Author information
Authors and Affiliations
Contributions
MD and SB conceived and designed research. ER, CM, CL and AMS conducted experiments. ER, MD, SB, AMS and AS analyzed data. ER, MD and SB wrote the manuscript. All authors read and approved the manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Nokwanda Pearl Makunga.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Rage, E., Marusic, C., Lico, C. et al. Optimisation of PD-FcY veterinary antigen secretion from Nicotiana benthamiana hairy roots and purification from the culture medium. Plant Cell Tiss Organ Cult 142, 23–39 (2020). https://doi.org/10.1007/s11240-020-01826-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-020-01826-1