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ORIGINAL RESEARCH article

Front. Plant Sci.
Sec. Plant Systems and Synthetic Biology
Volume 15 - 2024 | doi: 10.3389/fpls.2024.1407240
This article is part of the Research Topic Biomolecule Production in Plant Synthetic Biology View all articles

High Production of Recombinant Protein using Geminivirus-based Deconstructed Vectors in Nicotiana benthamiana

Provisionally accepted
Nan-Sun Kim Nan-Sun Kim 1Kyeong-Ryeol Lee Kyeong-Ryeol Lee 1*Jihyea Lee Jihyea Lee 1Eui-Joon Kil Eui-Joon Kil 2Juho Lee Juho Lee 1Seon-kyeong Lee Seon-kyeong Lee 1
  • 1 Department of Agricultural Biotechnology, National Institute of Agricultural Science (South Korea), Wanju, Republic of Korea
  • 2 Department of Plant Medicals, Andong National University, Andong, North Gyeongsang, Republic of Korea

The final, formatted version of the article will be published soon.

    We focused on the geminiviral vector systems to develop an efficient vector system for plant biotechnology. Begomoviruses and curtoviruses, which belong to the Geminiviridae family, contain an intergenic region (IR) and four genes involved in replication, including replication-associated protein (Rep, C1), transcriptional activator (TrAP, C2), and replication enhancer (REn, C3). Geminiviruses can amplify thousands of copies of viral DNA using plant DNA polymerase and viral replication-related enzymes and accumulate viral proteins at high concentrations. In this study, we optimized geminiviral DNA replicon vectors based on tomato yellow leaf curl virus (TYLCV), honeysuckle yellow vein virus (HYVV), and mild curly top virus (BMCTV) for the rapid, high-yield plant-based production of recombinant proteins. Confirmation of the optimal combination by codelivery of each replication-related gene and each IR harboring the Pontellina plumata-derived turbo green fluorescence protein (tGFP) gene via agroinfiltration in Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 3 days. Co-expression with the p19 protein of the tomato bush stunt virus, a gene-silencing suppressor, further enhanced tGFP accumulation by stabilizing mRNA. With this system, tGFP protein was produced at 0.7-1.2 mg/g leaf fresh weight, corresponding to 6.9-12.1% in total soluble protein. These results demonstrate the advantages of rapid and high-level production of recombinant proteins using the geminiviral DNA replicon system for transient expression in plants.

    Keywords: Viral vector, Geminivirus, transient expression, turbo green fluorescence protein, Nicotiana benthamiana

    Received: 26 Mar 2024; Accepted: 08 Jul 2024.

    Copyright: © 2024 Kim, Lee, Lee, Kil, Lee and Lee. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Kyeong-Ryeol Lee, Department of Agricultural Biotechnology, National Institute of Agricultural Science (South Korea), Wanju, 55365, Republic of Korea

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