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Endoplasmic reticulum-directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

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DOI

  • Thomas Beuchert Kallehauge, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. thoka@biosustain.dtu.dk.
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  • Stefan Kol, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
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  • Mikael Rørdam Andersen, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
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  • Christian Kroun Damgaard
  • Gyun Min Lee, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • ,
  • Helene Faustrup Kildegaard, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. hef@biosustain.dtu.dk.

When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide-dependent manner, to acquire specific post-translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER-directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER-directed mRNA, with no cytoplasmic translation of ER-directed recombinant proteins observed. These observations indicate that the recombinant mRNA, encoding ER-directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also recombinant protein trafficking between the ER and cytosol in CHO cells.

Original languageEnglish
JournalBiotechnology Journal
Volume11
Issue10
Pages (from-to)1362-1367
Number of pages6
ISSN1860-6768
DOIs
Publication statusPublished - 14 Sep 2016

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