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Updated: Jun 17, 2026

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Published on: October 16, 2014

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

Yanan Nan¹, Minglu Liu¹, Mingrui Yang¹

¹School of Life Science, Beijing University of Chinese Medicine.

Journal of Visualized Experiments : Jove

|June 15, 2026

Summary

This summary is machine-generated.

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Researchers developed an in vitro system to study how STING proteins are packaged into COPII vesicles for innate immunity signaling. This optimized method enhances reproducibility and scalability for analyzing ER export mechanisms.

Area of Science:

Cell Biology
Immunology
Molecular Biology

Background:

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is crucial for innate immunity.
STING, an endoplasmic reticulum (ER)-resident protein, must be transported via COPII vesicles to the Golgi apparatus to initiate signaling.
The precise mechanisms of STING sorting into COPII vesicles are not fully understood.

Purpose of the Study:

To establish an optimized in vitro workflow for reconstituting COPII vesicles.
To enable controlled analysis of cargo selection, specifically focusing on STING.
To provide a scalable and reproducible system for studying ER export.

Main Methods:

Developed a scalable method for high-concentration cytosol preparation from HEK-293F cells.

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Related Experiment Videos

Last Updated: Jun 17, 2026

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Published on: October 16, 2014

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

Published on: December 28, 2015

Established a procedure for generating semi-permeabilized HEK-293T cells as a defined membrane source.

Utilized Western blot analysis to validate COPII vesicle budding and STING cargo incorporation in vitro.

Main Results:

Achieved a basal STING packaging efficiency of approximately 20% into COPII vesicles under defined conditions.
Demonstrated the system's ability to manipulate biochemical parameters, such as nucleotide stimulation, to study their impact on cargo incorporation.
Validated the optimized workflow for analyzing COPII-mediated cargo sorting.

Conclusions:

The presented in vitro reconstitution system offers a robust and adaptable platform for investigating COPII-mediated cargo sorting.
This system facilitates the study of STING transport and can be extended to other transmembrane proteins and ER export factors.
The optimized workflow improves scalability, reduces cost, and enhances reproducibility compared to traditional cell-based methods.