Pohle, M., Curry, E. orcid.org/0000-0002-5123-8866, Holden, R. et al. (2 more authors) (2025) Synthetic G-quadruplex components for predictable, precise two-level control of mammalian recombinant protein expression. Nucleic Acids Research, 53 (14). gkaf732. ISSN: 0305-1048
Abstract
Control of mammalian recombinant protein expression underpins the in vitro manufacture and in vivo performance of all biopharmaceutical products. However, routine optimization of protein expression levels in these applications is hampered by a paucity of genetic elements that function predictably across varying molecular formats and host cell contexts. Herein, we describe synthetic genetic components that are specifically built to simplify bioindustrial expression cassette design processes. Synthetic G-quadruplex elements with varying sequence feature compositions were systematically designed to exhibit a wide range of regulatory activities and inserted into identified optimal positions within a standardized, bioindustry compatible core promoter-5′UTR control unit. The resulting library tuned protein production rates over two orders of magnitude, where DNA and RNA G-quadruplexes could be deployed individually or in combination to achieve synergistic two-level regulatory control. We demonstrate these components can predictably and precisely tailor protein expression levels in (i) varying gene therapy and biomanufacturing cell hosts and (ii) both plasmid DNA and synthetic messenger RNA contexts. As an exemplar use case, a vector design platform was created to facilitate rapid optimization of polypeptide expression ratios for difficult-to-express multichain products. Permitting simple, predictable titration of recombinant protein expression, this technology should prove useful for gene therapy and biopharmaceutical manufacturing applications.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | ©The Author(s) 2025. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Biochemistry and Cell Biology; Biological Sciences; Genetics; Biotechnology; Gene Therapy; G-Quadruplexes; Recombinant Proteins; Humans; Promoter Regions, Genetic; Animals; 5' Untranslated Regions; Plasmids; DNA; Genetic Vectors; RNA, Messenger; Gene Expression Regulation; HEK293 Cells |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > School of Chemical, Materials and Biological Engineering |
Funding Information: | Funder Grant number Engineering and Physical Sciences Research Council EP/T517835/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 06 Aug 2025 15:17 |
Last Modified: | 06 Aug 2025 15:17 |
Status: | Published |
Publisher: | Oxford University Press (OUP) |
Refereed: | Yes |
Identification Number: | 10.1093/nar/gkaf732 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:230140 |