Amplification of EBNA-1 through a single-plasmid vector-based gene amplification system in HEK293 cells as an efficient
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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Amplification of EBNA-1 through a single-plasmid vector-based gene amplification system in HEK293 cells as an efficient transient gene expression system Sun-Hye Park 1,2 & Jong-Ho Park 1,3 & Joo-Hyoung Lee 1,3 & Hoon-Min Lee 1,2 & Yeon-Ju Kang 1,2 & Eun-Ji Lee 1,2 & Seunghyeon Shin 3 & Gyun Min Lee 3 & Yeon-Gu Kim 1,2 Received: 30 July 2020 / Revised: 28 October 2020 / Accepted: 3 November 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Our previous work showed that there is a limitation in the use of dihydrofolate reductase (dhfr)/methotrexate (MTX)-mediated gene amplification systems in dhfr-non-deficient HEK293 cells, as endogenous dhfr may interfere with the amplification process. In the present study, we successfully generated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-amplified HEK293 cells in a dhfr-non-deficient HEK293 cell background using a single-plasmid vector-based gene amplification system with shRNA targeting the 3′-UTR of endogenous dhfr. The introduction of this shRNA efficiently downregulated the expression of endogenous dhfr in the HEK293 cells without affecting exogenous dhfr expression. The downregulation of endogenous dhfr improved the efficiency of EBNA-1 amplification, as evidenced by a comparison with the amplification extent in cells lacking shRNA expression at the same MTX concentration. The EBNA-1 expression levels from the EBNA-1-amplified clones selected in this study were higher than those obtained from EBNA-1-amplified clones that were generated using the conventional amplification in our previous study. Consistent with previous studies, EBNA-1 amplification improved the production of the Fc-fusion protein through a specific protein productivity (qp)-enhancing effect, rather than by improving cell growth or transfection efficiency. In addition, the N-glycan profiles in the Fc-fusion protein produced using this transient gene expression (TGE) system were not affected by EBNA-1 amplification. These results indicate the potential utility of EBNA-1-amplified mammalian cells, developed using a single-plasmid vector-based gene amplification system, for efficient protein production. Key points • EBNA-1-amplified HEK293 cells were established using gene amplification system. • EBNA-1 amplification in TGE system can increase the Fc-fusion protein productivity. • EBNA-1 amplification does not affect the N-glycan profile in the Fc-fusion protein. Keywords TGE . HEK293 cells . Gene amplification . shRNA . EBNA-1 . Fc-fusion protein Sun-Hye Park and Jong-Ho Park contributed equally to this work.
Introduction * Gyun Min Lee [email protected] * Yeon-Gu Kim [email protected] 1
Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, Korea
2
Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, Korea
3
Department of Bi
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