University of Guilan Caspian Journal of Environmental Sciences 1735-3033 24 2 2026 04 01 Molecular farming: Expression of recombinant Ocriplasmin in genetically transformed root culture of Nicotiana tabaccum 439 450 9602 10.22124/cjes.2026.9602 EN Ata Allah Sharafi Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Habibollah Samizadeh Lahiji Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Ali Sharafi Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran Farjad Rafeie Department of Agricultural Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Mohammad Javad Motamedi Molecular Biology Research Center, Green Gene Company, Tehran, Iran Journal Article 2026 05 13 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.

https://cjes.guilan.ac.ir/article_9602_0b05249259ce34546c1f25e71468c243.pdf