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

Summary

This protocol describes an optimized cell-free system for reconstituting STING incorporation into COPII vesicles, enabling controlled biochemical analysis of cargo sorting during ER export.

Abstract

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.

Introduction

The cGAS-STING signaling pathway serves as a fundamental mechanism of the innate immune system, sensing cytosolic double-stranded DNA (dsDNA) to initiate an antiviral response. Upon detecting dsDNA, the enzyme cGAS produces the cyclic dinucleotide cGAMP, which acts as a second messenger that binds to the STING^1,2. Crucially, STING resides as an inactive dimer in the endoplasmic reticulum (ER) under steady-state conditions³. Its activation requires rapid, highly regulated translocation from the ER to the Golgi apparatus. It is at the Golgi that STING recruits and activates the downstream kinases TBK1 and IRF3, leading to the production of type I interferons and pro-inflammatory cytokines⁴.

The transport of STING is mediated by COPII-coated vesicles, the universal vehicles for protein export from the ER⁵. The assembly of the COPII coat—comprising the small GTPase Sar1, the inner-coat Sec23/24 complex, and the outer-coat Sec13/31 complex—is responsible for both deforming the ER membrane into vesicles and selectively recruiting cargo^6,7. Sec22b is sorted into COPII vesicles via a structural epitope recognized by the Sec23/24 complex of COPII⁸. Other cargoes include the p24 family and the ERGIC marker ERGIC-53, both of which depend on a C-terminal dihydrophobic φC motif for sorting⁹. In contrast, certain ER-anchored proteins, such as ribophorin I (RPN1), remain confined to the ER and serve as markers of intact ER structures, as they are not transported via COPII vesicles¹⁰. Consequently, in cell-free COPII vesicle reconstitution experiments, Sec22b and p24 are often considered as COPII cargoes, while ribophorin serves as a marker to monitor ER contamination in vesicle preparations. While the general machinery of COPII transport is well-characterized, the specific molecular cues that trigger the selective sequestration of activated STING into these vesicles remain elusive. Understanding these sorting mechanisms is vital, as dysregulation of STING transport is linked to various autoimmune and autoinflammatory diseases, such as STING-associated vasculopathy with onset in infancy (SAVI) and COPA syndrome¹¹. However, studying these transient and highly dynamic events in intact cells remains technically challenging.

To address these challenges, an optimized cell-free reconstitution system was developed to interrogate the molecular requirements of COPII-mediated cargo sorting. While in vitro budding assays have long been a staple of cell biology¹², their widespread adoption is often limited by the high cost and complexity of preparing high-quality components. In this study, a streamlined workflow is introduced that utilizes HEK-293F suspension cells as an economical source of active cytosol and semi-permeabilized HEK-293T cells as a robust membrane donor. The overall workflow of the cell-free COPII vesicle reconstitution system is illustrated in Figure 1. In this system, Sec22b was used as a positive control to confirm cargo incorporation into vesicles, while RPN1 was utilized as a negative control to monitor and rule out ER membrane contamination. Using STING as a proof-of-concept cargo, this study demonstrated that the system effectively models essential biochemical aspects of STING sorting. This platform provided a versatile, scalable tool for identifying cytosolic factors and membrane signals that govern STING sorting, with potential applications for studying a wide array of other vesicular transport pathways.

Protocol

The HEK-293F (RRID: CVCL_6642) and HEK-293T (RRID: CVCL_0063) cell lines were obtained from the Cell Resource Center at Peking Union Medical College. Both cell lines were authenticated by short tandem repeat (STR) profiling and confirmed to be free of mycoplasma contamination. The reagents and the equipment used are listed in the Table of Materials.

No human participants or vertebrate animals were involved in this study. Therefore, ethical approval was not required. All experimental procedures were conducted in accordance with institutional biosafety guidelines.

1. Preparation of HEK-293F cell cytosol

1. Seed HEK-293F cells at a density of 1 × 10⁶ cells/mL in 100 mL defined mammalian culture medium supplemented with 1% penicillin–streptomycin. Culture the cells in a 500 mL conical flask.
2. Incubate the suspension culture at 37 °C with 5% CO₂ in a shaking incubator for 60 h.
3. Harvest the cells by centrifugation at 300 × g for 5 min at 4 °C. Determine the total cell number using an automated cell counter.
4. Wash the cell pellet with 50 mL ice-cold phosphate-buffered saline (PBS). Centrifuge at 300 × g for 5 min at 4 °C.
5. Resuspend the cell pellet in an equal volume of ice-cold B88 buffer (20 mM HEPES, pH 7.3; 250 mM sorbitol; 150 mM potassium acetate; 5 mM magnesium acetate).
6. Lyse the cells on ice by passing the suspension through a 5 mL syringe fitted with a 22-gauge needle for more than 100 strokes.
7. Examine cell lysis by light microscopy. Remove cell debris by centrifugation at 20,000 × g for 10 min at 4 °C.
8. Transfer the supernatant to ultracentrifuge tubes. Centrifuge at 100,000 × g for 1 h at 4 °C.
   CAUTION: Ensure ultracentrifuge tubes are properly balanced to prevent equipment damage and safety hazards.
9. Collect the clarified supernatant. Concentrate the sample using a 3 kDa molecular weight cutoff (MWCO) ultrafiltration device at 4,000 × g at 4 °C.
10. Measure protein concentration using a bicinchoninic acid (BCA) assay. Adjust the concentration to 30–40 mg/mL.
    CAUTION: Handle liquid nitrogen with appropriate protective equipment to avoid cryogenic injury.
11. Aliquot the cytosol. Snap-freeze the aliquots in liquid nitrogen and store at −80 °C (Figure 2A)
    CAUTION: Avoid more than two freeze–thaw cycles. Store aliquots at −80 °C for up to 6 months.

2. Preparation of semi-permeabilized HEK-293T cells

1. Plate HEK-293T cells expressing full-length human STING at a density of 3 × 10⁶ cells per 15 cm dish in 15 mL DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Incubate at 37 °C with 5% CO₂ for 48 h.
2. Harvest the cells at approximately 60% confluency.
3. Aspirate the medium and wash the cells twice with 10 mL ice-cold PBS.
4. Add 2 mL of 0.25% trypsin–EDTA. Incubate at room temperature for 2–3 min to detach the cells.
5. Add 200 µL soybean trypsin inhibitor (1 mg/mL) to stop trypsinization. Resuspend the cells in 10 mL ice-cold KHM buffer (20 mM HEPES, pH 7.2; 110 mM potassium acetate; 2 mM magnesium acetate).
6. Pellet the cells by centrifugation at 250 × g for 3 min. Discard the supernatant.
   CAUTION: Digitonin is toxic. Handle in a fume hood with appropriate personal protective equipment.
7. Resuspend the cells in 6 mL ice-cold KHM buffer. Add 6 µL digitonin stock solution (40 mg/mL in DMSO).
8. Mix gently by inversion. Incubate on ice for 5 min.
9. Add 8 mL KHM buffer to dilute digitonin. Centrifuge at 250 × g for 3 min to collect permeabilized cells.
10. Resuspend the pellet in 14 mL ice-cold HEPES buffer (50 mM HEPES, pH 7.3; 90 mM potassium acetate). Incubate on ice for 10 min.
11. Centrifuge at 250 × g for 3 min. Resuspend the pellet in 1 mL ice-cold KHM·Cl buffer (20 mM HEPES, pH 7.3; 110 mM KCl; 2 mM MgCl₂).
12. Centrifuge at 10,000 × g for 15 s. Discard the supernatant.
13. Resuspend the pellet in 50 µL KHM·Cl buffer. Measure protein concentration using a BCA assay and adjust to 5–7 mg/mL.

3. Cell-free COPII vesicle reconstitution experiment

1. Prepare all reagents listed in the Table of Materials. Aliquot and store at −80 °C.
2. Prepare a 50 µL reaction mixture containing 110 mM KCl, 20 mM HEPES (pH 7.2), 2 mM MgCl₂, protease inhibitor cocktail (1×), 0.2 mM GTP, and an ATP regeneration system (40 mM creatine phosphate, 0.2 mg/mL creatine phosphokinase, 1 mM ATP).
3. Add cGAMP to a final concentration of 10 µM. Incubate the reaction at room temperature for 15 min.
4. Add 50 µg semi-permeabilized HEK-293T cells. Mix gently.
5. Add 80 µg HEK-293F cytosol to initiate vesicle formation. Incubate at 37 °C for 45 min.
6. Place the reaction on ice to terminate budding. Centrifuge at 12,000 × g for 20 min at 4 °C.
7. Collect the supernatant containing vesicles. Retain the pellet containing organelles.
8. Ultracentrifuge the supernatant at 100,000 × g for 1 h at 4 °C. Resuspend the vesicle pellet in 30 µL protein loading buffer.
9. Heat samples at 95 °C for 5 min. Centrifuge at 20,000 × g for 5 min to remove precipitates.
10. Perform SDS-PAGE and transfer proteins to PVDF membranes. Probe with antibodies against Sec22b, STING, and RPN1.

Results

During the preparation of HEK-293F cell cytosol, two parameters were evaluated to assess cytosol quality. HEK-293F cells were cultured at high density, yielding cytosolic extracts with high protein concentrations. In addition, the extent of cell disruption following syringe-based lysis was assessed by light microscopy, confirming efficient cell lysis under the applied conditions (Figure 2B).

For the generation of semi-permeabilized HEK-293T cells, cultures were ha...

Discussion

The reconstitution of essential biochemical reactions in vitro has laid the foundation for cell-free systems, which have been instrumental in numerous landmark discoveries. These include the identification of cGAS as a cytosolic DNA sensor^14,15, the elucidation of the role of cytochrome c in apoptosis, and the discovery of the first histone demethylase, JHDM1^16,17. Such defined biochemical approa...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 mL sterile syringes	WEGO	V526273	Used for mechanical cell lysis
Adenosine 5′-triphosphate (ATP)	MCE	HY-B2176	100 mM stock in water, pH 7.3, stored at −80 °C
Adobe Illustrator	Adobe Systems Incorporated	SCR_010279	Used for creating conceptual illustrations and schematic diagrams
ATP regeneration system (10×)	—	—	Energy-regenerating mixture containing creatine phosphate, creatine kinase, and ATP
BCA Protein Assay Kit	Thermo Fisher	23227	Colorimetric protein assay
BioRender	BioRender	SCR_018361	Used for creating conceptual illustrations and schematic diagrams
BSA	Biorigin	BN20815	Blocking reagent
Carbon dioxide shaking incubator	ZhiChu	ZCZY-CS8	Maintains temperature, CO2, and agitation for suspension culture
CO2 incubator	Thermo Fisher	3110	Maintains temperature and CO2 for adherent culture
Conventional microscope	Olympus	CX43	Used to assess cell lysis and morphology
Creatine kinase	Roche	CKRO	20 mg/mL stock in buffer, stored at −80 °C
Creatine phosphate	Roche	CRPHO-RO	Component of ATP regeneration system
Desktop centrifuge	Eppendorf	5810R	Used for low-speed centrifugation
Digitonin	Sigma	D5628	40 mg/mL stock in DMSO, stored at −20 °C
DMEM	Corning	10-013-CMR	Growth medium for HEK-293T cells
D-Sorbitol	Sangon	A100691	1 M stock solution in water, stored at 4 °C
ECL detection reagent	Biorigin	BN16009	Chemiluminescent substrate
Electrophoresis tank	Bio-Rad	1658001	Apparatus for SDS-PAGE
Fetal bovine serum (FBS)	Corning	35-015-CV	Serum supplement
Fixed-angle rotor (TFT 80.2)	Thermo Fisher	54456	Rotor for ultracentrifugation
FreeStyle 293-F cells	Thermo Fisher	R79007	Used for cytosol preparation
Goat anti-mouse IgG (H+L)	Invitrogen	31430	HRP-conjugated secondary antibody
Goat anti-rabbit IgG (H+L)	Invitrogen	31460	HRP-conjugated secondary antibody
GraphPad Prism	GraphPad Software	SCR_002798	Used for performing statistical analysis (one-way ANOVA) and generating the quantitative bar graph in Figure 4D.
Guanosine 5′-triphosphate (GTP)	MCE	HY-113225	10 mM stock in water, pH 7.3, stored at −80 °C
HEK-293T cells expressing human STING	—	—	Generated in-house; used as membrane source
HEPES buffer (1 M, pH 7.3)	Beyotime	C0215	Buffer component for solution preparation
Imaging system	Bio-Rad	12003153	Used for signal detection
Magnesium acetate tetrahydrate	Hampton Research	HR2-561	Buffer component for B88 preparation
Magnesium chloride hexahydrate	Hampton Research	HR2-559	Buffer component
Microcentrifuge	Eppendorf	5420R	Used for medium-speed centrifugation
Microcentrifuge	Eppendorf	5420	Used for routine centrifugation
NanoDrop spectrophotometer	Thermo Fisher	840-317400	Used for protein quantification
OPM-CD Trans293 medium	OPM	P82019	Serum-free medium for HEK-293F suspension culture
Penicillin–streptomycin (100×)	Servicebio	G4003	Antibiotic supplement for cell culture
Phenylmethyl sulfonyl fluoride (PMSF)	Sangon	A610425	Protease inhibitor
Phosphatase inhibitors	Lablead	C0104	Prevent protein dephosphorylation
Phosphate-buffered saline (PBS)	Servicebio	G4202	Buffer for washing cells
Potassium acetate	Sigma	236497	1 M stock solution in water, stored at 4 °C
Power supply	Bio-Rad	1645052	Provides voltage for electrophoresis
Protease inhibitor cocktail, EDTA-free	Roche	4693132001	Broad-spectrum protease inhibitor mixture
PVDF membrane	Millipore	ISEQ00010	Membrane for Western blotting
RPN1 antibody (rabbit mAb)	Abcam	ab197888	ER marker
SDS-PAGE gel (4–20%)	—	—	Used for protein separation
SDS-PAGE loading buffer (5×)	Solarbio	P1040	Protein sample preparation buffer
Sec22b antibody (rabbit mAb)	Abcam	ab181076	COPII cargo marker
Soybean trypsin inhibitor (SBTI)	Sigma	T9128	1 mg/mL stock in PBS, stored at −20 °C
STING antibody (rabbit mAb)	CST	13647	Primary antibody
TBST buffer	Servicebio	G0004	Washing buffer for immunoblotting
Thermal cycler	Bio-Rad	186-1096	Used for heating and incubation
Thermostatic metal bath	DLAB	5062102100	Maintains constant temperature
Transfer cell	Bio-Rad	1703930	Used for protein transfer
Trypsin-EDTA (0.25%)	Servicebio	G4011	Used for cell detachment
Ultracentrifuge	Thermo Fisher	75000100	Used for high-speed centrifugation
Ultracentrifuge tubes (2 mL)	Thermo Fisher	54460	Tubes compatible with ultracentrifugation
Ultrafiltration tube (4 mL, 10 kDa MWCO)	Millipore	UFC8010	Used for cytosol concentration