Development of A Cell-Free COPII Vesicle Reconstitution Protocol For Investigating STING Sorting In A HEK-293...

The cGAS-STING pathway is a central component of innate immunity. Upon activation, the endoplasmic reticulum (ER)-resident protein STING is packaged into COPII vesicles and transported to the Golgi apparatus to initiate downstream signaling. Despite the importance of this process, the molecular mechanisms governing STING sorting into COPII vesicles remain incompletely understood. Here, an optimized workflow is presented for the in vitro reconstitution of COPII vesicles to facilitate controlled analysis of cargo selection. A scalable method is described for preparing high-concentration cytosol (30-40 mg/mL) from HEK-293F suspension cells, combined with a straightforward procedure for generating semi-permeabilized HEK-293T cells as a defined membrane source. Compared to traditional adherent cell-based systems, this approach improves scalability, reduces cost, and enhances reproducibility. Using STING as a model cargo, COPII vesicle budding and cargo incorporation are validated by Western blot analysis, with a basal packaging efficiency of approximately 20% under defined conditions. This system allows controlled manipulation of biochemical parameters, including nucleotide stimulation, to assess their effects on cargo incorporation. This protocol provides a robust and adaptable platform for investigating COPII-mediated cargo sorting and can be extended to study additional transmembrane proteins and regulatory factors involved in ER export.