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Large-Scale Production of Recombinant Noggin and R-Spondin1 Proteins Required for the Maintenance of Stem Cells in Intestinal Organoid Cultures.
Journal article

Large-Scale Production of Recombinant Noggin and R-Spondin1 Proteins Required for the Maintenance of Stem Cells in Intestinal Organoid Cultures.

  • Hacker DL Protein Production and Structure Core Facility (PPSCF), École Polytechnique Fédérale de Lausanne (EPFL), School of Life Sciences, Lausanne, Switzerland. david.hacker@epfl.ch.
  • Ordóñez-Morán P Division of Cancer and Stem Cells, School of Medicine, Centre for Cancer Sciences, Biodiscovery Institute, University of Nottingham, Nottingham, UK.
  • 2020-07-25
Published in:
  • Methods in molecular biology (Clifton, N.J.). - 2020
Affinity chromatography
HEK293
Intestinal organoids
Orbital shaking
Polyethylenimine
Recombinant protein
Stem cells
Transfection
mNoggin
mR-Spondin1
English The presence of the proteins mouse R-Spondin1 (mRSpo1) and mouse Noggin (mNoggin) in a 3D-organoid culture allows for the maintenance of intestinal stem cells. Here, we describe a transient gene expression method for the production of these proteins from human embryo kidney 293 (HEK293) cells cultivated in suspension using orbitally shaken bioreactors. Plasmid DNA was delivered into cells using the cationic polymer polyethylenimine (PEI). The 7-day production cultures were performed in the presence of valproic acid (VPA), an enhancer of recombinant gene expression. Both proteins were secreted from the transfected cells. mRSpo1 was produced as a secreted Fc fusion protein (mRSpo1-Fc) and purified by protein A-based affinity chromatography. mNoggin was produced as a secreted histidine-tagged protein (mNoggin-His) and purified by immobilized metal affinity chromatography (IMAC). This transient transfection system supports a high production efficiency.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/112051
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