MMP-3 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-MMP3
In stock
- SKU
- REC-MMP3
Target Overview
MMP-3 (Stromelysin-1; UniProt P08254; EC 3.4.24.17) is a zinc-dependent endopeptidase of the matrix metalloproteinase family encoded by the MMP3 gene. The full-length human protein spans 477 amino acids and comprises an N-terminal signal peptide, propeptide domain, catalytic domain containing the conserved HEXXHXXGXXH zinc-binding motif, and a C-terminal hemopexin-like domain. This recombinant form is produced in HEK293 cells, a mammalian expression system that supports native-like glycosylation and folding, making it well suited for enzymatic activity studies where post-translational modifications may influence substrate engagement or inhibitor binding. MMP-3 carries a broad substrate repertoire, cleaving fibronectin, laminin, type I/III/IV/V gelatins, collagens III, IV, IX, and X, and cartilage proteoglycans. It also functions as a proenzyme activator — notably for MMP-9 — and processes growth factors and plasminogen within the extracellular matrix (ECM). In solution, the proenzyme form is activated by the plasmin cascade; researchers studying activation kinetics routinely use recombinant proMMP-3 alongside plasmin or APMA to establish defined activation conditions before downstream assays. Beyond secreted ECM functions, published data document intracellular and nuclear roles for MMP-3, including HTRA2-mediated activation under cellular stress conditions and antiviral activity mediated through NF-κB pathway modulation. This recombinant is used in fluorogenic substrate cleavage assays (e.g., Mca-RPKPVE-Nval-WRK(Dnp)-NH₂), inhibitor IC50 determinations, and as a positive control antigen for antibody validation. Researchers validating anti-MMP-3 antibodies can pair this recombinant with the matched Triple Point Biologics antibody (SKU: RP-MMP3) to confirm band identity by Western blot or antigen specificity by ELISA. Validated reactivity covers human protein; cross-reactivity with mouse, rat, monkey, dog, and cat is predicted based on sequence conservation.
Background
Applications
- Fluorogenic substrate cleavage activity assay (e.g., Mca-RPKPVE-Nval-WRK(Dnp)-NH₂) to confirm enzymatic activity lot-to-lot
- Inhibitor IC50 determination for small-molecule or peptide-based MMP-3 inhibitors in microplate format
- Proenzyme activation kinetics study using APMA or plasmin to characterise the propeptide removal step
- Positive control antigen for Western blot and ELISA validation alongside matched Triple Point anti-MMP-3 antibody (RP-MMP3)
- ELISA standard curve generation for quantification of MMP-3 in conditioned medium or synovial fluid samples
- Substrate specificity profiling by mass spectrometry to map cleavage sites in ECM protein panels
- MMP-9 zymogen activation assay, using recombinant MMP-3 as the upstream activating protease
- Molecular docking and SPR/BLI binding validation for candidate MMP-3 inhibitor compounds
References
- Zhang L et al. Effect of needle knife on autophagy and matrix degradation of chondrocytes in rabbit knee osteoarthritis. Zhongguo Gu Shang. 2026. doi:10.12200/j.issn.1003-0034.20241126. PMID: 42338210.
- Xu R et al. Combining Network Pharmacology, Machine Learning, Molecular Docking, and Experimental Validation to Explore the Mechanism of Danggui-Shaoyao-San in treating Rheumatoid arthritis. Curr Pharm Des. 2026. doi:10.2174/0113816128431656260305144620. PMID: 42337901.
- Sun HL et al. Evaluating the Therapeutic Potential of Hyriopsis cumingii Polysaccharide in Osteoarthritis: Insights from a Mouse Model. Rejuvenation Res. 2026. doi:10.1177/15491684251414069. PMID: 42333691.
- Takahata K et al. Novel Biomaterial-Based Synovial Fluid Analysis Reveals Protective microRNA Signatures in a Mouse Model of Acute Synovitis-Driven Osteoarthritis. FASEB J. 2026. doi:10.1096/fj.202505006R. PMID: 42328732.
- Hong S et al. Vaginal dysbiosis and inflammatory signatures in preterm labor: an integrated model for predicting preterm birth. Front Immunol. 2026. doi:10.3389/fimmu.2026.1809046. PMID: 42327783.
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 | Secreted; Extracellular matrix |
Frequently Asked Questions
What molecular weight band should I expect for recombinant MMP-3 on SDS-PAGE or Western blot?
The full-length MMP-3 proenzyme (477 aa) runs at ~54 kDa under reducing SDS-PAGE conditions. Because this recombinant is produced in HEK293 cells with native-like glycosylation, the observed band typically migrates slightly higher than the predicted unmodified molecular weight of ~51.3 kDa. If you have activated the enzyme by propeptide removal, expect a catalytic-domain fragment near ~28 kDa and a hemopexin domain near ~22 kDa. When using RP-MMP3 for Western blot validation, the proenzyme band at ~54 kDa is the most reliable reference.
What processing forms and isoforms of MMP-3 does this recombinant correspond to?
This product corresponds to the canonical human MMP-3 proenzyme (UniProt P08254-1, 477 aa), expressed without the signal peptide but retaining the propeptide domain, catalytic domain, and C-terminal hemopexin-like domain. It is supplied in the latent zymogen form; the cysteine-switch mechanism keeps the active site occluded until propeptide removal. Activation can be achieved with APMA (1 mM, 37°C, 1 h) or via autoactivation at low pH. This is the predominant secreted isoform relevant to ECM remodeling, tumor invasion, and osteoarthritis research.
What substrates does recombinant MMP-3 cleave, and what is the preferred fluorogenic substrate for activity assays?
MMP-3 has a broad substrate repertoire in vivo — fibronectin, laminin, collagens III/IV/IX/X, cartilage proteoglycans, and plasminogen — and it activates pro-MMP-9. For in vitro activity assays, the fluorogenic peptide Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (MMP-3 substrate I) is widely used; cleavage is monitored at Ex/Em 320/405 nm. A typical assay uses 100–500 nM activated recombinant MMP-3 in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35) with 10–20 µM substrate. Confirm activation before assay by pre-incubating with 1 mM APMA.
What buffer conditions are optimal for recombinant MMP-3 enzymatic activity assays?
The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol — is compatible with direct use in activity assays after dilution. For assay, dilute into 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35 (omitting glycerol at the working dilution). CaCl2 is essential for catalytic zinc coordination; omitting it abolishes activity. Keep pH between 7.0 and 7.8 — MMP-3 shows a sharp activity drop below pH 6.5. Avoid EDTA or EGTA, as chelation of zinc or calcium will irreversibly inactivate the enzyme.
What starting concentration of recombinant MMP-3 should I use for IC50 inhibitor profiling experiments?
For IC50 determinations, we recommend activating the proenzyme first (1 mM APMA, 37°C, 1 h), then using 50–200 nM active MMP-3 in the final assay volume. Keep enzyme concentration well below the Km of your fluorogenic substrate (~10 µM) to maintain initial-rate conditions and avoid substrate depletion artifacts. Include 1 mM PMSF controls to define background, and use a reference inhibitor (e.g., GM6001 at 10 nM) to confirm assay window stability across plate reads. Titrate inhibitor in at least 8 half-log steps to obtain reliable curve fits.
Can I use recombinant MMP-3 as a positive control for Western blot with the RP-MMP3 antibody?
Yes — this is one of the primary intended uses. REC-MMP3 and RP-MMP3 are produced and validated in-house as a matched pair. Load 20–50 ng of REC-MMP3 per lane alongside your cell lysate or conditioned medium samples. Under reducing conditions, the proenzyme produces a clean band at ~54 kDa that the RP-MMP3 rabbit polyclonal detects with high sensitivity. This approach confirms antibody functionality, correct band identity, and transfer efficiency simultaneously. RP-MMP3 has been validated for Western blot; see /anti-mmp-3-rabbit-polyclonal-antibody for recommended dilutions and blocking conditions.
How much recombinant MMP-3 should I load for a Western blot positive control lane?
20–50 ng per lane is sufficient for a strong, clean signal with RP-MMP3 under standard ECL detection. If you are using a higher-sensitivity substrate (e.g., femtomolar-range HRP substrates), 5–10 ng may be adequate and will reduce background. Denature in Laemmli buffer with 100 mM DTT at 95°C for 5 min before loading; partial reduction can cause smearing near the 54 kDa region. Run on a 10–12% polyacrylamide gel for best resolution of the proenzyme versus any activated fragments that may appear in your experimental lanes.
How should I store and handle recombinant MMP-3 to preserve enzymatic activity long-term?
REC-MMP3 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol and is stable at -20°C for at least 12 months from the date of receipt when stored in single-use aliquots. Repeated freeze-thaw cycles cause measurable activity loss — plan aliquot sizes around your typical experiment. On thawing, keep on ice and use within 4–6 hours; avoid leaving at room temperature. Do not dilute into buffers lacking CaCl2 for extended periods. If activity appears diminished, first verify activation state before assuming degradation.
Validation imagery coming soon
Western blot validation figures for REC-MMP3 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.