Endoplasmic reticulum-directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells.
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.
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. thoka@biosustain.dtu.dk.
Thomas Beuchert
TB
Kallehauge
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Stefan
S
Kol
Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
Mikael
M
Rørdam Andersen
Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
Christian
C
Kroun Damgaard
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.|Department of Biological Sciences, KAIST, Daejeon, Korea.
Gyun Min
GM
Lee
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. hef@biosustain.dtu.dk.
Helene
H
Faustrup Kildegaard
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics*; D004921:Erythropoietin / Q000235:genetics*; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000069283:Rituximab / Q000235:genetics*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D004921:Erythropoietin; D000069283:Rituximab
CHO cells; Endoplasmic reticulum; Fractionation; Translocation; mRNA