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Bacterial Artificial Chromosome-based Protein Expression Platform Using the Tol2 Transposon System

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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.

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Acknowledgements

This research was supported by Research Assistance Program (2021) in the Incheon National University. This research was also supported by priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A0304195411).

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Authors and Affiliations

  1. Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, 22012, Korea

    Myeong Uk Kuk, Ji Yun Park, Eun Seon Song, Haneur Lee, Yun Haeng Lee, Junghyun Joo, Hyung Wook Kwon & Joon Tae Park

  2. Convergence Research Center for Insect Vectors, Incheon National University, Incheon, 22012, Korea

    Myeong Uk Kuk, Ji Yun Park, Eun Seon Song, Haneur Lee, Yun Haeng Lee, Junghyun Joo, Hyung Wook Kwon & Joon Tae Park

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  1. Myeong Uk Kuk
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Contributions

MUK, HWK, and JTP conceived of and designed the experiments. MUK, JYP, ESS, HL, YHL, and JJ performed the experiments. MUK analyzed the data. MUK, HWK, and JTP wrote and edited the manuscript.

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Correspondence to Hyung Wook Kwon or Joon Tae Park.

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Kuk, M.U., Park, J.Y., Song, E.S. et al. Bacterial Artificial Chromosome-based Protein Expression Platform Using the Tol2 Transposon System. Biotechnol Bioproc E 27, 344–352 (2022). https://doi.org/10.1007/s12257-021-0222-y

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