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aGPCR-HEK: A Stable High-Expression Inducible Mammalian Cell Expression System for Adhesion GPCR Structural Biology Applications
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DOI：10.21769/BioProtoc.5621.png)
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Abstract
En 中文
ADGRL4 is an adhesion G protein-coupled receptor (aGPCR) implicated in multiple tumours. In our experience, conventional insect cell-based baculovirus expression systems have not yielded sufficient correctly folded ADGRL4 protein for purification and cryo-electron microscopy (cryo-EM) analysis. Here, we describe aGPCR-HEK, a six-week protocol that tagged aGPCRs. The method comprises lentiviral production in Lenti-X 293T cells, transduction of target adherent HEK293S GnTI- TetR cells, flow cytometry enrichment of uninduced GFP-positive cells displaying leaky expression, adaptation to suspension culture, and large-scale tetracycline induction and harvesting of cells for downstream purification and cryo-EM. The system yields reproducible, milligram-scale quantities of folded aGPCR suitable for structural and biochemical studies.
Keywords:
Adhesion GPCR (aGPCR)
HEK293S GnTI-TetR
Lentivirus
Stable cell line
Tetracycline induction
Inducible expression
Journal
B
IF:
1.1
Papers:
191
Citations:
5.4K
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Cited Papers