ArticleBio-protocol2026
aGPCR-HEK: A Stable High-Expression Inducible Mammalian Cell Expression System for Adhesion GPCR Structural Biology Applications.
Article in Bio-protocol, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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2 authors.
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Abstract
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 establishes stable tetracycline-inducible mammalian HEK293S GnTI- TetR cell lines expressing N-terminally HA- and GFP-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. Key features • Establishes a stable, tetracycline-inducible HEK293S GnTI- TetR expression system in adherent or suspension cells for high-level expression of N-terminally HA- and GFP-tagged aGPCRs. • Uses lentiviral transduction for efficient aGPCR genomic integration and employs a tetracycline-repressor enabling high-level expression following tetracycline induction. • Uses flow cytometry enrichment of leaky GFP-positive cells to enhance inducibility and yield. • Provides suspension culture adaptation and large-scale tetracycline induction for structural biology-grade protein production suitable for purification and cryo-EM analysis.
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