Microsoft, Portal and Ragon Institute Team Up to Build AI-Powered Map of Immune System
Microsoft, Portal and Ragon Institute Team Up to Build AI-Powered Map of Immune System
Portal mechanoporation delivers Promega’s LgBiT protein directly into live cells, enabling real-time measurement of protein degradation, target engagement, and phosphorylation — without cell lysis. In PROTAC validation assays, this approach showed near-complete luminescence signal reduction at 10 µM LC-2, consistent with substantial protein degradation, with full dose-response curves generated entirely in intact, living cells.
The HiBiT/LgBiT system is Promega’s binary NanoLuc technology for detecting endogenous protein levels inside cells. HiBiT is a small 11-amino-acid peptide tag that can be genetically fused to any protein of interest via CRISPR knock-in. LgBiT is a large (18 kDa) complementary protein. When HiBiT and LgBiT combine inside a cell, they reconstitute an active NanoLuc luciferase that produces a bright luminescent signal proportional to the amount of tagged protein present.
The challenge is getting LgBiT protein into cells. At 18 kDa, LgBiT cannot cross the cell membrane on its own. Traditional approaches rely on cell lysis — breaking cells open so LgBiT can access HiBiT-tagged proteins in solution. This works for endpoint measurements but destroys the living system, eliminating the ability to perform kinetic measurements, track real-time degradation, or study drug responses in physiologically relevant conditions.
Mechanoporation solves this by delivering LgBiT protein directly into the cytosol of intact, living cells. Cells pass through precisely engineered constrictions in a MicroBooster cartridge, creating transient openings in the cell membrane that allow LgBiT to enter. The cell membrane reseals rapidly, trapping the protein inside a fully functional cell.
Portal mechanoporation delivers LgBiT protein into live cells through a three-step physical process that requires no viral vectors, lipid nanoparticles, or electrical current.
Step 1 — Combine cells and cargo: Cells expressing HiBiT-tagged endogenous proteins are suspended with purified LgBiT protein in solution.
Step 2 — Mechanical membrane disruption: The cell suspension flows through a MicroBooster cartridge containing precisely engineered pores. As cells transit these pores, controlled mechanical stress creates transient openings in the cell membrane.
Step 3 — Intracellular delivery and recovery: LgBiT protein in the surrounding solution diffuses through the transient membrane pores directly into the cytosol. The cell membrane reseals rapidly, trapping LgBiT inside the cell where it can complement with endogenous HiBiT-tagged proteins to produce luminescence.
This method preserves cell viability and native biology. Unlike electroporation, which causes transcriptional disruption across hundreds of genes, mechanoporation causes zero gene misregulation at q<0.05, maintaining the cell’s native signaling pathways and making downstream assay results more physiologically representative.
Yes. Portal mechanoporation enables quantitative, dose-dependent measurement of PROTAC-mediated protein degradation in living cells using the HiBiT/LgBiT complementation system.
In validation experiments, LgBiT protein was delivered into cells expressing a HiBiT-tagged endogenous protein. The PROTAC degrader LC-2 was then added at increasing concentrations, and relative luminescence was measured after 24 hours of treatment.
0 µM (vehicle control): 100% luminescence — Baseline
1 µM: ~42% luminescence — 55%+ reduction
5 µM: ~25% luminescence — 75%+ reduction
10 µM: ~10% luminescence — 85%+ signal reduction
The assay produced a clear, dose-dependent degradation curve with 85%+ signal reduction at 10 µM LC-2, consistent with near-complete protein degradation. Because the cells remain alive throughout the experiment, this approach enables capabilities that lysate-based methods cannot provide: kinetic measurements at multiple time points from the same population, real-time tracking of degradation onset and recovery, and assessment of protein degradation under physiologically relevant conditions with intact cellular machinery.
Portal-enabled live-cell detection achieves comparable signal to lysate-based methods while preserving the biological context that lysis destroys.
In a direct comparison using Promega’s Lumit immunoassay for phosphorylated BTK (pBTK), Portal mechanoporation delivered four antibodies simultaneously into live cells. Pervanadate stimulation was used to induce BTK phosphorylation, and luminescence was measured in both live-cell (Portal) and lysate conditions.
Untreated: ~350,000 lysate / ~150,000 Portal — 40%+ of lysate
Pervanadate-stimulated: ~1,400,000 lysate / ~1,100,000 Portal — 75%+ of lysate
Fold induction: 4x lysate / ~7x Portal — Higher dynamic range
Two findings stand out. First, Portal achieved 75%+ of lysate signal intensity in stimulated cells — demonstrating that live-cell antibody delivery produces sufficient signal for quantitative protein detection. Second, Portal showed an approximately 7-fold signal induction compared to 4-fold for lysate, indicating higher dynamic range in the live-cell format. This is likely because intact cells maintain active signaling pathways that enhance phosphorylation responses, while lysis disrupts these pathways and dilutes the signal.
The simultaneous delivery of four antibodies in a single mechanoporation step also demonstrates that multiplexed intracellular detection is practical, not just theoretical.
Beyond protein degradation monitoring, Portal mechanoporation enables a suite of Promega intracellular assays that previously required either lysate-based workflows or cell-permeable probe workarounds.
NanoBRET measures direct drug-target binding inside living cells using bioluminescence resonance energy transfer. The assay requires delivery of a fluorescent tracer molecule that is normally cell-impermeable. Portal delivers this tracer directly into the cytosol, enabling competitive displacement measurement as drug molecules outcompete the tracer for target binding.
Ponatinib: Src kinase — ~85%+ reduction (~20→~3 mBu)
Bosutinib: Src kinase — ~90%+ reduction (~25→~2 mBu)
Both kinase inhibitors produced clear dose-dependent displacement curves in NanoLuc-Src cells, confirming that mechanoporation-delivered tracers faithfully report intracellular target engagement.
The Lumit immunoassay detects post-translational modifications (phosphorylation, ubiquitination) using paired antibodies that produce luminescence when in close proximity on a target protein. Portal delivers these antibody pairs directly into living cells, enabling detection of phosphorylation events in real time without lysis.
Portal’s partnership with Promega extends across the NanoBit, NanoBRET, and Lumit product families. Because mechanoporation is cargo-agnostic — delivering proteins, antibodies, and small-molecule tracers with the same workflow — researchers can access Promega’s full intracellular assay suite without developing custom cell-permeable probe variants for each application.
Mechanoporation-enabled intracellular assays provide three practical advantages for drug discovery teams working on PROTACs, molecular glues, and other modalities where cell permeability limits early-stage screening.
Skip probe permeabilization development. Traditional approaches require engineering cell-permeable versions of intracellular probes — a process that can take months and require significant investment per probe. Mechanoporation delivers the native, unmodified probe directly, eliminating this development step entirely.
Enable “Direct-to-Biology” validation. Portal’s mechanoporation decouples molecule permeability from biological activity during drug discovery. This means researchers can test biochemical hits directly inside living cells before investing in medicinal chemistry to optimize permeability — potentially avoiding up to a year and $1 million in medicinal chemistry optimization by identifying inactive compounds before committing to permeability engineering.
Scale from discovery to high-throughput screening. The same mechanoporation principle works across Portal’s product line: the Gateway system for research-scale assay development and the Galaxy platform for automated 96-well and 384-well screening. Galaxy integrates as a single cartridge addition to existing liquid handling robots, requiring no new capital equipment and no change to dispensing speed.
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