ADAM-17 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-ADAM17
In stock
- SKU
- REC-ADAM17
Target Overview
ADAM-17 (Disintegrin and metalloproteinase domain-containing protein 17; UniProt P78536) is a transmembrane zinc-dependent metalloprotease encoded by the ADAM17 gene on human chromosome 2p25. The full-length human protein spans 824 amino acids and contains an N-terminal signal peptide, prodomain, catalytic metalloprotease domain (bearing the HExxHxxGxxH zinc-binding motif), disintegrin-like domain, cysteine-rich domain, epidermal growth factor-like domain, transmembrane anchor, and short cytoplasmic tail. This recombinant (REC-ADAM17) is produced in HEK293 mammalian cells, which supports the glycosylation and disulfide-bond formation required for native-like conformation and proteolytic activity. The HEK293 expression system is preferred over prokaryotic hosts for ADAM-family sheddases precisely because correct folding of the catalytic and disintegrin domains is essential for substrate engagement. Researchers use this recombinant across several experimental contexts. In enzymatic activity assays, it cleaves fluorogenic peptide substrates derived from known shedding targets such as TNF-α, TGF-α, and IL-6R, enabling kinetic characterisation (Km, kcat, kcat/Km). In inhibitor screening formats, it serves as the enzyme component in fluorescence-based IC50 assays against hydroxamate-class and other metalloprotease inhibitors. In substrate identification studies, it is incubated with candidate ectodomain constructs and cleavage products analysed by SDS-PAGE or mass spectrometry. The recombinant also functions as a positive-control antigen for antibody validation: researchers working with the matched Triple Point Biologics antibody (RP-ADAM17) can use REC-ADAM17 as a defined loading standard on Western blots or as a coating antigen in ELISA-based specificity panels. Because ADAM17 cleaves more than a dozen cell-surface proteins relevant to inflammation, haematopoiesis, and neurobiology, a well-characterised recombinant with demonstrated activity is a routine requirement in labs studying shedding pathway regulation.
Background
Applications
- Fluorogenic substrate cleavage assay using TNF-α or TGF-α peptide substrates (Km, kcat determination)
- Inhibitor IC50 determination for hydroxamate-class and small-molecule metalloprotease inhibitors in fluorescence-based formats
- Ectodomain shedding reconstitution: incubation with IL-6R, LAG3, or ACE2 ectodomain constructs followed by SDS-PAGE or LC-MS/MS cleavage-site mapping
- Antibody validation positive control on Western blot — use alongside matched Triple Point Biologics antibody RP-ADAM17
- ELISA coating antigen for antibody specificity and cross-reactivity panels
- iRhom2/ADAM-17 regulatory pathway studies: cell-free shedding reconstitution with recombinant substrate ectodomains
- Substrate selectivity profiling against a panel of synthetic peptides by FRET-based or mass spectrometry readout
- SPR or BLI binding kinetics determination for anti-ADAM-17 therapeutic candidate antibodies or inhibitor scaffolds
References
- Flodbring Larsson P et al. Targeting oncogenic TβRI signaling inhibits androgen-independent prostate cancer growth and metastasis. Signal Transduct Target Ther. 2026. doi:10.1038/s41392-026-02737-x PMID: 42303991
- Kaur A et al. Platelet TREM-Like Transcript-1 (TLT-1) regulation in healthy donors and patients at risk of bleeding and thrombosis. J Thromb Haemost. 2026. doi:10.1016/j.jtha.2026.06.009 PMID: 42297145
- São Pedro RB et al. Screening of autoinflammatory genes in patients with SARS-CoV-2-associated MIS-C. Hum Genomics. 2026. doi:10.1186/s40246-026-00985-0 PMID: 42277935
- Hannemann C et al. Deficiency of Inactive Rhomboid Protein 2 (iRhom2) Attenuates Macrophage Atherogenicity. Biology (Basel). 2026. doi:10.3390/biology15110860 PMID: 42274511
- Gao Z et al. Identification and multicenter validation of a 4-gene plasma signature for early recognition and risk assessment in hypertensive intracerebral hemorrhage. Genomics. 2026. doi:10.1016/j.ygeno.2026.111268 PMID: 42218946
Additional Specifications
| Storage Buffer | 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol |
|---|---|
| Endotoxin Level | <0.1 EU/µg by LAL |
| Purity (%) | >90% by SDS-PAGE |
| Expression System | HEK293 |
| Subcellular Localization | Type I transmembrane; cell surface; Golgi → endosomes |
Frequently Asked Questions
What molecular weight band should I expect for recombinant ADAM-17 on SDS-PAGE or Western blot?
The full-length human ADAM-17 (UniProt P78536) has a predicted molecular weight of ~93 kDa, but the mature processed form — after removal of the signal peptide and prodomain — runs at approximately 85 kDa under reducing SDS-PAGE conditions. Because REC-ADAM17 is produced in HEK293 cells, N-linked glycosylation typically shifts the apparent MW to ~100–130 kDa on a gel. If you see a diffuse band in that range, glycosylation heterogeneity is the most likely explanation, not degradation. Under non-reducing conditions, disulfide-linked species may run higher.
What form of ADAM-17 does REC-ADAM17 correspond to — zymogen, prodomain-cleaved, or mature ectodomain?
REC-ADAM17 corresponds to the catalytically active ectodomain fragment, expressed without the transmembrane anchor or cytoplasmic tail. The prodomain is removed during production to yield the mature, active metalloprotease form. This mirrors the shed ectodomain detected in conditioned media and serum. It retains the catalytic domain (HExxHxxGxxH zinc-binding motif), disintegrin-like domain, cysteine-rich domain, and EGF-like domain. If your experiment requires zymogen-form or prodomain-containing ADAM-17, this product is not the appropriate choice.
What substrates does ADAM-17 cleave and which is best for a fluorescent activity assay?
ADAM-17's best-characterized physiological substrates include TNF-α (cleavage at Ala76-Val77), pro-HB-EGF, TGF-α, and amphiregulin. For in vitro fluorescent activity assays, the synthetic FRET peptide Mca-PLAQAV-Dpa-RSSSR-NH2 (derived from the TNF-α cleavage site) is the most widely validated substrate for ADAM-17. Use it at 10–25 µM final concentration. Excitation/emission is typically 320/405 nm. Include 1 mM PMSF or 10 µM marimastat as a negative control to confirm ADAM-17-specific signal. EDTA (>1 mM) will chelate catalytic zinc and abolish activity.
What buffer conditions should I use for ADAM-17 activity assays and is the storage buffer compatible?
REC-ADAM17 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, and 5 mM CaCl2. This buffer is directly compatible with most fluorescent substrate assays. For optimal activity, run reactions at pH 7.4–8.0 in the presence of 5 mM CaCl2 and 0.05–0.1% Brij-35 or Triton X-100 to minimize non-specific surface adsorption. Avoid EDTA, EGTA, or 1,10-phenanthroline — all will inhibit the zinc-dependent catalytic mechanism. Glycerol from the storage buffer at low carry-over volumes (≤5% final) does not measurably affect Km or Vmax in published assay formats.
What starting concentration of recombinant ADAM-17 should I use for an IC50 inhibitor screening assay?
For IC50 determination using the Mca-PLAQAV-Dpa-RSSSR-NH2 FRET substrate, a starting enzyme concentration of 1–5 nM REC-ADAM17 in a 100 µL reaction volume is typical. Use substrate at or below the Km (~10 µM) to maximize inhibitor sensitivity and avoid substrate depletion artifacts within the assay window (30–60 min at 37°C). Titrate inhibitor across 8–10 concentrations spanning at least 3 log units. Pre-incubate enzyme with inhibitor for 15–30 minutes before initiating the reaction with substrate. Run at least two independent experiments in triplicate for statistically reliable IC50 values.
Can I use REC-ADAM17 as a Western blot positive control for the matched RP-ADAM17 antibody?
Yes — this is one of the primary intended uses for REC-ADAM17 alongside the matched rabbit polyclonal antibody RP-ADAM17 (/anti-adam-17-rabbit-polyclonal-antibody). Both products originate from the same TPB production pipeline, and RP-ADAM17 is validated against REC-ADAM17 in Western blot, ensuring a confirmed reactive band. Load 50–100 ng of REC-ADAM17 per lane on a 4–12% Bis-Tris gel under reducing conditions; expect a glycosylated band at ~100–130 kDa. This pairing is particularly useful when validating RP-ADAM17 against a new cell lysate where endogenous ADAM-17 expression level is uncertain.
How much recombinant ADAM-17 should I load as a positive control and what band should RP-ADAM17 detect?
Load 50–100 ng of REC-ADAM17 per lane. With RP-ADAM17 used at a 1:1,000–1:2,000 dilution (standard for HRP-conjugated secondary detection), you should obtain a clear band in the ~100–130 kDa range reflecting the glycosylated ectodomain. At 200+ ng loading, non-specific smearing can occur; lower amounts (25–50 ng) are workable if using a sensitive chemiluminescent substrate. Because RP-ADAM17 was raised against the ADAM-17 ectodomain and validated against this recombinant over TPB's 32 years of proteinase antibody production, cross-reactivity with non-specific bands at the indicated dilution range is minimal.
How should I handle, dilute, and store REC-ADAM17 to maintain proteolytic activity over time?
REC-ADAM17 is supplied in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. Store at -20°C; the glycerol content prevents freeze damage at this temperature. Avoid repeated freeze-thaw — each cycle can reduce activity by 10–20%. On the day of use, thaw on ice and dilute immediately into your assay buffer containing CaCl2. Carrier protein (0.1% BSA) helps prevent adsorption losses at low working concentrations (<10 nM). Working dilutions should be used within 4 hours at 4°C; do not refreeze diluted material. Opened aliquots stored at 4°C are stable for up to 48 hours with no significant activity loss.
Validation imagery coming soon
Western blot validation figures for REC-ADAM17 will be published here as they are produced in-house.
If you would like to see existing validation data for this antibody before publication, request a sample copy.