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Cited by 38Viral Infectious Diseases and Gene Expression in InsectsRNA and protein synthesis mechanismsProtein purification and stability

Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

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Eisenhut, Peter, Mebrahtu, Aman, Moradi Barzadd, Mona, Thalén, Niklas, Klanert, Gerald, Weinguny, Marcus, Sandegren, Anna, Su, Chao +3 more·2020·Nucleic Acids Research
DOI 10.1093/nar/gkaa847PMC PMC7672427
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Abstract

Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed ‘regulation elements (RgE)’, in the 5′-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5′-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5′-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

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  • An extensively stable secondary structure with high GC content in the 5′ UTR, particularly in the 5′-proximal region, tends to decrease translation efficiency.

    “It is well established that an extensively stable secondary structure with high GC content in the 5′ UTR -particularly in the 5′-proximal region -tends to decrease translation efficiency [10,34].”

    From Identification of novel 5’ UTR variants for enhanced mRNA translation and vaccine immunogenicity (2026) · cites this paper as [34]

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Authors
Eisenhut, P., Mebrahtu, A., Moradi Barzadd, M., Thalén, N., Klanert, G., Weinguny, M., Sandegren, A., Su, C., Hatton, D., Borth, N., & Rockberg, J.
Affiliations
  • Austrian Centre of Industrial Biotechnology (Austria) (AT)
  • KTH Royal Institute of Technology (SE)
  • KTH Royal Institute of Technology, Department of Protein Science, 10691 Stockholm, Sweden
  • Affibody (Sweden) (SE)
  • SOBI AB, Tomtebodavägen 23A, Stockholm, Sweden
  • AstraZeneca (United Kingdom) (GB)
Corresponding author
  • Borth, Nicole
  • Rockberg, Johan
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2020-10-13
Volume
48
Issue
20
Pages
e119-e119
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkaa847
PMC
PMC7672427
OpenAlex
W3092177332
Cited by
38
References
2
Citations by year
2026: 3 · 2025: 7 · 2024: 10 · 2023: 8 · 2022: 7 · 2021: 3
FWCI
1.44
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Austrian Centre of Industrial Biotechnology
  • BMVIT
  • BMDW
  • SFG
  • Standortagentur Tirol
  • Austrian Science Fund (W1224)
  • VINNOVA
  • CellNova
  • AAVNova
  • AdBioPro
  • Swedish Foundation for Strategic Research (SB16-0017)
  • Novo Nordisk Foundation (NNF10CC1016517)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
5' untranslated region, RNA hairpin, translation efficiency, protein expression regulation, recombinant protein expression, CHO cells, IgG heavy chain, SUMF1, arylsulfatase A, helper factor, protein aggregation, fluorescent reporter
MeSH terms
Animals, Cerebroside-Sulfatase, Cricetulus, Gene Expression Regulation, Genetic Vectors, Humans, Immunoglobulin G, Nucleic Acid Conformation, Recombinant Proteins, Protein Biosynthesis, Protein Engineering, Gene Expression, CHO Cells, 5' Untranslated Regions, Oxidoreductases Acting on Sulfur Group Donors, Inverted Repeat Sequences, HEK293 Cells

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