Biotechnology and Bioprocess Engineering 2022; 27(3): 344-352  
Bacterial Artificial Chromosome-based Protein Expression Platform Using the Tol2 Transposon System
Myeong Uk Kuk, Ji Yun Park, Eun Seon Song, Haneur Lee, Yun Haeng Lee, Junghyun Joo, Hyung Wook Kwon, and Joon Tae Park
Myeong Uk Kuk, Ji Yun Park, Eun Seon Song, Haneur Lee, Yun Haeng Lee, Junghyun Joo, Hyung Wook Kwon*, Joon Tae Park*
Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Korea
Convergence Research Center for Insect Vectors, Incheon National University, Incheon 22012, Korea
Tel: +82-32-835-8090; Fax: +82-32-835-0754
E-mail: hwkwon@inu.ac.kr
Tel: +82-32-835-8841; Fax: +82-32-835-0754
E-mail: joontae.park@inu.ac.kr
Received: August 6, 2021; Revised: November 16, 2021; Accepted: December 10, 2021; Published online: June 30, 2022.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Conventional vector systems, including plasmid-based vectors, are mainly used in mammalian cells for the production of biopharmaceuticals. Plasmid-based vectors express transgene by the random integration of recombinant transgenes into the genome. Transgene expression is greatly influenced by the surrounding chromatin, and in most cases, expression is weak and tends to be suppressed over time. Therefore, a novel strategy is required to create clones that maintain increased transgene expression. In this study, we used a bacterial artificial chromosome (BAC) containing the Rosa26 locus, which allows constitutive and ubiquitous gene expression. Moreover, we improved the Rosa26 BAC-mediated expression system by incorporating the Tol2 transposon system with helper vector, resulting in improved efficiency of protein production and maintained productivity even in single-cell clones. Furthermore, the recombinant Rosa26 BAC was improved in terms of protein productivity by using helper mRNA instead of helper vector. Finally, establishment of an optimal molar ratio between the recombinant Rosa26 BAC and helper mRNA helped to achieve maximal protein productivity. Taken together, our results provide an effective strategy for improving BAC-based expression systems for biopharmaceutical production.
Keywords: Rosa26 BAC, Tol2 transposon system, helper mRNA, bacterial artificial chromosome

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