Overview

Inspired by the regulated secretion mechanisms of neurons and pancreatic β-cells, we designed a rapid-response, drug-inducible protein secretion switch based on the chemically induced dissociation of NS3a and ANR¹.

Our favorite composite part, P_CMV-IgK-(Nluc-FS)₃-TMD-(NS3a)₂-BGHPA (BBa_25WEPAKX), forms the core secretion module of our system. It functions together with the anchoring module P_CMV-ANR-mCherry-LifeAct-BGHPA (BBa_25NYLBFF) to build chemSPARK, a modular secretion control platform (See our Design and Results page).

Under normal conditions, the NS3a-ANR interaction anchors secretory vesicles to the actin cytoskeleton, preventing release. When cells are exposed to Grazoprevir, an FDA-approved NS3a inhibitor, the drug disrupts the NS3a-ANR complex, triggering immediate vesicle release and rapid protein secretion (Figure 1).

Figure 1.Schematic illustration of the secretion ON system mediated by Grazoprevir-induced NS3a-ANR dissociation.(TMD, transmembrane domain).

Methods

Cell Culture and Transfection

HEK293T cells (ATCC CRL-3216) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C and 5% CO₂. Cells were seeded at 5 × 10⁴ per well in 24-well plates and transfected 12 h later using a PEI-based protocol (PEI:DNA = 5:1, w/w). HEK293T cells were co-transfected with pNC25092 (P_CMV-IgK-(Nluc)₃-TMD-(NS3a)₂) and either pNC25086 (P_CMV-LifeAct-mCherry; control) or pNC25091 (P_CMV-ANR-LifeAct).The transfection mix was replaced with fresh medium 6 h post-transfection. Transfection was performed at 16 h after seeding 5 × 10⁴ mammalian cells into each well of a 24-well plate. The cell culture medium was replaced with fresh medium (not containing transfection reagents) at 6 h after transfection. HEK-293T cells were transfected using a PEI-based protocol at a PEI:DNA ratio of 5:1 (w/w) and in a transfection volume of 50 μL native serum-free DMEM per well.

Reporter Quantification

Secreted NanoLuc levels were measured from cell culture supernatants using the Nano-Glo Luciferase Assay System (Promega, N1120). All measurements were performed in biological quadruplicates (n = 4). Statistical analyses used two-tailed unpaired t-tests (P<0.05 considered significant).

Result

To test the secretion switch, HEK293T cells were co-transfected with plasmids carrying P_CMV-IgK-(Nluc-FS)₃-TMD-(NS3a)₂-BGHPA (BBa_25WEPAKX) and plasmids carrying P_CMV-ANR-mCherry-LifeAct-BGHPA (BBa_25NYLBFF) at a 1:3 DNA ratio (100 ng : 300 ng per well).

After 48 h, cells were treated with 10 µM Grazoprevir (experimental group) or DMSO (control). Supernatants were collected 2 h post-treatment for NanoLuc quantification. Cells treated with Grazoprevir showed a significant increase in NanoLuc secretion, confirming that NS3a-ANR dissociation releases pre-anchored vesicles and activates rapid protein secretion (Figure 2).

Figure 2.NanoLuc levels in culture supernatants 2 h after Grazoprevir (10 µM) or DMSO treatment. Data shown as mean ± SD (n = 4); ***P<0.001.

Dynamic Response

To assess activation kinetics, HEK293T cells were co-transfected with plasmids carrying P_CMV-IgK-(Nluc-FS)₃-TMD-(NS3a)₂-BGHPA (BBa_25WEPAKX) and plasmids carrying P_CMV-ANR-mCherry-LifeAct-BGHPA (BBa_25NYLBFF) at a 1:3 DNA ratio (100 ng : 300 ng per well).. Cells were then treated with 10 µM Grazoprevir or DMSO and supernatants were collected at a series of different time points within 24 hours post-treatment.

NanoLuc secretion rose rapidly within the first hour and peaked around 3 h, reaching an approximate 40-fold induction relative to untreated controls (Figure 3).

These results demonstrate that the secretion switch responds dynamically to drug input, achieving full activation within hours.

Figure 3.Time-course of NanoLuc secretion following Grazoprevir stimulation (10 µM). Mean ± SD, n = 3.

Dose-Dependent Response

To characterize dose sensitivity, cells were treated with a gradient of Grazoprevir concentrations (0-10 µM) 48 h after transfection. Supernatants were collected 2 h post-stimulation for NanoLuc measurement. The system exhibited graded, dose-dependent activation, with secretion increasing steadily from 100 nM and peaking at 10 µM Grazoprevir, where a maximum 40-fold induction was observed (Figure 4).

Figure 4.Dose-dependent secretion response to Grazoprevir treatment (0-10 µM). Data presents mean ±SD, n = 3 independent experiments, fitted to a symmetric sigmoid curve.

While NanoLuc serves as the reporter in our prototype, it can be readily replaced with user-defined therapeutic or functional proteins, allowing teams to adapt the system to diverse applications. For example, the secretion module could be reprogrammed to release insulin or pramlintide for diabetes therapy², atrial natriuretic peptide (ANP) for cardiac function regulation³, or phytohormones for controlled agricultural biomanufacturing⁴.

The composite part P_CMV-IgK-(Nluc-FS)₃-TMD-(NS3a)₂-BGHPA serves as the core output module of our chemSPARK system. It enables rapid, tunable, and reversible control of protein secretion in mammalian cells through drug-induced protein dissociation.

This part demonstrates the feasibility of building post-translational gene switches that respond to clinically relevant small molecules, offering a powerful foundation for therapeutic protein delivery, synthetic cell communication, and dynamic regulation in mammalian synthetic biology.