Molecular farming: Expression of recombinant Ocriplasmin in genetically transformed root culture of Nicotiana tabaccum

Document Type : Research Paper

Authors

1 Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran

2 Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran

3 Molecular Biology Research Center, Green Gene Company, Tehran, Iran

10.22124/cjes.2026.9602

Abstract

Ocriplasmin, also known as microplasmin (JetreaTM), is a novel recombinant drug approved by the FDA. It is a truncated human plasmin used for the treatment of vitreoretinopathies. In the present study, ocriplasmin was expressed in Nicotiana tabacum hairy roots as a molecular farming platform. The gene of this recombinant protein was designed, synthesized and sub-cloned from pUC57 to plant expression vector pBI121 at the XbaI and the SacI sites. Then, the Agrobacterium rhizogenes strains MSU440, A4 and ATCC15834 were transformed by the obtained plasmid. For plant transformation and hairy root induction, tobacco seeds were cultivated in MS culture medium for 4 weeks at a phytotron. Leaf pieces were infected by the abovementioned A. rhizogenes strains. The induction of hairy roots and the co-transformation was confirmed by molecular analysis. The highest hairy root induction percentage, 92% were found in A. rhizogenes strain ATCC15834. After confirming the presence of the gene of interest by PCR, total protein was extracted from the obtained transgenic roots and the recombinant protein was purified by the Ni-NTA column due to the presence of His-tag. The obtained proteins were subjected to SDS-PAGE, ELISA and Western blot tests. According to the results, 56 μg mL-1 of recombinant protein were found in the purified protein solution based on the Bradford method. The total protein content across three lines of hairy roots was measured at 67.2 μg. The highest total protein concentration and recombinant protein yield were determined to be 61 μg mL-1 and 1732 μg mL-1, respectively. Based on our results, we suggest that tobacco transgenic roots may be suitable sources for the production of this human protein.
 

Keywords

References

Aerts, F, Noppen, B, Fonteyn, L, Derua, R, Waelkens, E, de Smet, MD & Vanhove, M 2012, Mechanism of inactivation of ocriplasmin in porcine vitreous. Biophysical Chemistry, 165-166: 30-38.
Badrhadad, A, Nazarian-Firouzabadi & F, Ismaili, A 2018, Fusion of a chitin-binding domain to an antibacterial peptide to enhance resistance to Fusarium solani in tobacco (Nicotiana tabacum). 3 Biotech, 8: 391.
Bharathi, JK, Suresh, P, Samy, Prakash, MA & Muneer, S 2024, Exploring recent progress of molecular farming for therapeutic and recombinant molecules in plant systems. Heliyon, 10: e37634.
Beebe, DC 2015, Understanding the adverse effects of ocriplasmin. JAMA Ophthalmology, 133: 229.
Benz, MS, Packo, KH, Gonzalez, V, Pakola, S, Bezner, D, Haller, JA & Schwartz, SD 2010, A placebo-controlled trial of microplasmin intravitreous injection to facilitate posterior vitreous detachment before vitrectomy. Ophthalmology, 117: 791-7.
Bhisitkul, RB 2001, Anticipation for enzymatic vitreolysis. British Journal of Ophthalmology, 85: 1-2.
Bradford, MM 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.
Caizhen, G, Yan, G, Ronron, C, Lirong, Y, Panpan, C, Yuanbiao, Q & Qingshan, L 2015, Zirconium phosphatidylcholinebased nanocapsules as an in vivo degradable drug delivery system of MAP30, a momordica anti-HIV protein. International Journal of Pharmaceutics, 483: 188-199.
Cardon, F, Pallisse, R, Bardor, M, Caron, A, Vanier, J, Ele Ekouna, JP, Lerouge, P, Boitel‐Conti, M & Guillet, M 2019, Brassica rapa hairy root-based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase. Plant Biotechnology Journal, 17: 505-516.
de Smet, MD, Gandorfer, A, Stalmans, P, Veckeneer, M, Feron, E, Pakola, S & Kampik, A 2009, Microplasmin intravitreal administration in patients with vitreomacular traction scheduled for vitrectomy: the MIVI I trial. Ophthalmology, 116: 1349-1355.
de Smet, MD, Valmaggia, C, Zarranz-Ventura, J & Willekens, B 2009, Microplasmin: ex vivo characterization of its activity in porcine vitreous. Investigative Ophthalmology & Visual Science, 50: 814-819.
Fahim, AT, Khan, NW & Johnson, MW 2014, Acute panretinal structural and functional abnormalities after intravitreous ocriplasmin injection. JAMA Ophthalmology, 132: 484-486.
Fischer, R & Buyel, JF, Molecular farming - the slope of enlightenment. Biotechnology Advances, 40: 107519.
Freund, KB, Shah, SA & Shah, VP 2013, Correlation of transient vision loss with outer retinal disruption following intravitreal ocriplasmin. Eye (London), 27: 773-774.
Gandorfer, A, Rohleder, M, Sethi, C, Eckle, D, Welge-Lussen, U, Kampik, A, Luthert, P & Charteris, D 2004, Posterior vitreous detachment induced by microplasmin. Investigative Ophthalmology & Visual Science, 45: 641-647.
Gutierrez-Valdes, N, Häkkinen, ST, Lemasson, C, Guillet, M, Oksman-Caldentey, KM, Ritala, A & Cardon, F 2020, Hairy root cultures a versatile tool with multiple applications. Frontiers in Plant Science, 11: 33.
Han, DP, Abrams, GW & Aaberg, TM 1988, Surgical excision of the attached posterior hyaloid. Archives of Ophthalmology, 106: 998-1000.
Hoelscher, MP, Forner, J, Calderone, S, Krämer, C, Taylor, Z, Loiacono, FV, Agrawal, S, Karcher, D, Moratti, F, Kroop, X & Bock, R 2022, Expression strategies for the efficient synthesis of antimicrobial peptides in plastids. Nature Communications, 13: 5856.
Holaskova, E, Galuszka, P, Frebort, I & Oz, MT 2015, Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology. Biotechnology Advances, 33: 1005-1023.
Khademi, M, Varasteh-Shams, M, Nazarian-Firouzabadi & F, Ismaili, A 2020, New recombinant antimicrobial peptides confer resistance to fungal pathogens in tobacco plants. Frontiers in Plant Science, 11: 1236.
Khademi, M, Nazarian-Firouzabadi, F, Ismaili, A & Shirzadian Khorramabad, R 2019, Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco. MicrobiologyOpen, 8: e837.
Kumari, S & Tennakoon, I, Molecular farming: implication for future pharmaceutical products. Sri Lankan Journal of Health Sciences, 1: 51.
Laemmli, UK 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
Liu H & Timko MP 2022, Improving protein quantity and quality - the next level of plant molecular farming. International Journal of Molecular Sciences, 23: 1326.
Mirmazloum, I, Slavov, AK & Marchev, AS 2024, The untapped potential of hairy root cultures and their multiple applications. International Journal of Molecular Sciences, 25(23), 12682. https://doi.org/10.3390/ ijms252312682.
Moon, KB, Park, JS, Park, YI, Song, IJ, Lee, HJ, Cho, HS, Jeon, JH & Kim, HS 2020 Development of systems for the production of plant-derived biopharmaceuticals. Plants (Basel), 9: 30.
Murashige, T & Skoog, FA 1962, A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15: 473-497.
Nagai, N, Demarsin, E, Van Hoef, B, Wouters, S, Cingolani, D, Laroche, Y & Collen, D 2003, Recombinant human microplasmin: production and potential therapeutic properties. Journal of Thrombosis and Haemostasis, 1: 307-313.
Niknejad, A 2018, Plant-based expression systems for protein and antimicrobial peptide production. Nova Biologica Reperta, 5: 262-73.
Noppen, B, Fonteyn, L, Aerts, F, De Vriese, A, De Maeyer, M, Le Floch, F, Barbeaux, P, Zwaal, R, Vanhove, M, Autolytic degradation of ocriplasmin: A complex mechanism unraveled by mutational analysis. Protein Engineering, Design and Selection, 27: 215-223.
Richards, E, Reichardt, M & Rogers, S 1994, Preparation of genomic DNA from plant tissue. Current Protocols in Molecular Biology, 27: 231-237.
Ron, M, Kajala, K, Pauluzzi, G, Wang, D, Reynoso, MA, Zumstein, K, Garcha, J, Winte, S, Masson, H & Inagaki, S 2014, Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiology, 166: 455-469.
Russell, DW & Sambrook, J 2001, Molecular cloning: a laboratory manual, vol 1. Cold Spring Harbor Laboratory Cold Spring Harbor, NY.
Sebag J 1988, Pharmacologic vitreolysis. Retina, 18: 1-3.
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. Molecular Biotechnology, 61: 241-252.
Siridewa K 2021, Molecular pharming: a new approach for a healthy future by a vast development in the pharmaceutical industry. Current Trends in Biotechnology and Pharmacy, 15: 315-324.
Stefanini, FR, Maia, M, Falabella, P, Pfister, M, Niemeyer, M, Kashani AH, Humayun MS, Koss MJ 2014, Profile of ocriplasmin and its potential in the treatment of vitreomacular adhesion. Clinical Ophthalmology, 8: 847-856.
Tibbetts, MD, Reichel, E & Witkin, AJ. Vision loss after intravitreal ocriplasmin: correlation of spectral-domain optical coherence tomography and electroretinography. JAMA Ophthalmology, 132: 487-490.
Trese, MT 2002, Enzymatic-assisted vitrectomy. Eye (London), 16: 365-368.
Trese, MT 2000, Enzymatic vitreous surgery. Seminars in Ophthalmology, 15: 116-121.
Vander, JF & Kleiner, R 1992, A method for induction of posterior vitreous detachment during vitrectomy. Retina, 12: 172-173.
Webster, DE & Thomas, MC 2012, Post-translational modification of plant-made foreign proteins; glycosylation and beyond. Biotechnology Advances, 30: 410-418.
Wilken, LR & Nikolov, ZL 2012, Recovery and purification of plant-made recombinant proteins. Biotechnology Advances, 30: 419-433.
Caspian Journal of Environmental Sciences
Volume 24, Issue 2
April 2026
Pages 439-450

Files

Share

How to cite

Statistics