| Autor: |
David L. Hacker, Paloma Ordóñez-Morán |
| Rok vydání: |
2020 |
| Předmět: |
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| Zdroj: |
Methods in Molecular Biology ISBN: 9781071607466 |
| DOI: |
10.1007/978-1-0716-0747-3_10 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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