MMP-25 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-MMP25
In stock
- SKU
- REC-MMP25
Target Overview
MMP-25 (Matrix metalloproteinase-25, UniProt Q9NPA2) is a 562-amino-acid glycosylphosphatidylinositol (GPI)-anchored membrane-type matrix metalloproteinase (MT-MMP) classified under EC 3.4.24.-. Also designated MT6-MMP or Leukolysin, it is expressed at the cell membrane and is documented to promote activation of progelatinase A (pro-MMP-2), placing it upstream in the pericellular proteolytic cascade that remodels the extracellular matrix. This recombinant is produced by transient expression in HEK293 cells, which provides mammalian glycosylation and folding appropriate for an MT-MMP family member. The full-length human sequence spans 562 residues; the expressed construct covers the catalytic ectodomain suitable for in-vitro biochemical work. HEK293-derived material is particularly relevant for investigators studying GPI-anchored MMP biology, as the eukaryotic expression system preserves the disulfide architecture of the catalytic and hemopexin domains. Researchers use this recombinant in several experimental contexts: measuring proteolytic activity against fluorogenic or gelatin-based substrates, determining inhibitor potency (IC50 / Ki) in small-molecule screens, and serving as a defined positive-control antigen for antibody validation by Western blot or ELISA. Validated species reactivity for this target is human; cross-reactivity with non-human primate and other pan-mammalian orthologs is predicted based on sequence conservation but has not been independently confirmed under these production conditions. Investigators validating anti-MMP-25 antibodies can pair this recombinant with the matched Triple Point Biologics antibody reagent (SKU: RP-MMP25), which has been validated for Western blot applications — providing a consistent antigen–antibody system within a single supplier.
Background
Applications
- Proteolytic activity assay against fluorogenic peptide substrates (e.g., Mca-PLGL-Dpa-AR-NH2 or gelatin-based substrates)
- Pro-MMP-2 (progelatinase A) activation assay in defined cell-free systems
- Inhibitor IC50 / Ki determination in small-molecule or peptide-based MMP-25 inhibitor screens
- Positive-control antigen for Western blot validation of anti-MMP-25 antibodies (pair with RP-MMP25)
- ELISA capture or detection standard for MMP-25 quantification assays
- Substrate cleavage profiling by mass spectrometry to characterise MMP-25 cleavage-site specificity
- Cell-based ECM remodelling studies using exogenous recombinant enzyme to supplement or compare endogenous activity
- Biophysical binding assays (SPR, ITC) to characterise interactions between MMP-25 and candidate inhibitors or endogenous TIMPs
References
- San Sebastian-Jaraba I et al. Metalloproteinase 25 promotes abdominal aortic aneurysm and atherosclerosis. Atherosclerosis. 2026. doi:10.1016/j.atherosclerosis.2026.120676. PMID: 41713388.
- Wu T et al. From bioinformatic identification to functional validation: MMP25 as a pro-inflammatory mediator in osteoarthritis. J Orthop Surg Res. 2026. doi:10.1186/s13018-026-06732-4. PMID: 41673749.
- Zhao ZH et al. Expression of key genes in IVIG-unresponsive Kawasaki disease and their immune correlation based on bioinformatics analysis. Immunol Res. 2026. doi:10.1007/s12026-026-09746-6. PMID: 41575683.
- Zou M et al. Forced and voluntary exercise exert differential neuroprotective effects in cerebral ischemia-reperfusion injury by inhibiting neutrophil infiltration and blood-brain barrier disruption. Brain Res Bull. 2026. doi:10.1016/j.brainresbull.2025.111668. PMID: 41344617.
- Wang X et al. NR5A2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting MMP25 transcription. Biochim Biophys Acta Mol Basis Dis. 2026. doi:10.1016/j.bbadis.2025.168121. PMID: 41297641.
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-25 on SDS-PAGE or Western blot?
The full-length MMP-25 precursor is predicted at ~63 kDa from its 562-aa sequence (UniProt Q9NPA2), but HEK293-expressed material carries mammalian N-linked glycosylation, so the apparent MW on reducing SDS-PAGE typically runs between 65–72 kDa. Under non-reducing conditions the band may shift slightly due to intact disulfide bonds in the catalytic and hemopexin domains. When using this recombinant as a positive control on Western blot alongside antibody RP-MMP25, expect the primary band in the 65–72 kDa window.
Is recombinant MMP-25 the full-length protein or just the catalytic domain, and has the signal peptide been removed?
The expressed construct covers the catalytic ectodomain of human MMP-25 and is produced in HEK293 cells, which process the N-terminal signal sequence during secretion. The GPI-anchor signal at the C-terminus is not included in this soluble recombinant, making it suitable for in-vitro biochemical work without membrane tethering. The prodomain is removed during activation, yielding the mature catalytic form. This matches the biologically relevant processing state documented for MT6-MMP/Leukolysin in leukocyte and tumor microenvironment studies.
What substrates does recombinant MMP-25 cleave and which fluorogenic peptide substrate should I use for an activity assay?
MMP-25 (EC 3.4.24.-) cleaves extracellular matrix components including gelatin and fibronectin, and is documented to activate progelatinase A (pro-MMP-2) pericellularly. For fluorogenic activity assays, the generic MMP substrate Mca-PLGL-Dpa-AR-NH2 (Knight substrate) is widely used across MT-MMP family members and is compatible with MMP-25. Assay in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂ — matching the storage buffer — to avoid dilution-related buffer mismatch. Monitor fluorescence at Ex/Em 320/405 nm.
What buffer and calcium concentration should I use when setting up a recombinant MMP-25 activity assay in vitro?
The recombinant is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol — a buffer specifically formulated for metalloproteinases requiring divalent cation coordination at the active-site zinc. For activity assays, dilute directly into the same buffer omitting glycerol if assay volume allows, keeping CaCl₂ at 5–10 mM. Avoid EDTA or EGTA-containing buffers, as calcium chelation will abolish catalytic activity. Including 0.05% Brij-35 can reduce non-specific surface adsorption at low enzyme concentrations.
What starting concentration of recombinant MMP-25 should I use for an IC50 inhibitor assay?
For IC50 determinations, a typical starting point is 0.5–2 nM active enzyme with substrate held at or near its Km (commonly 10–20 µM for fluorogenic peptides). At these concentrations, the Morrison tight-binding correction may be unnecessary unless your inhibitor Ki approaches sub-nanomolar range. Run a substrate conversion check first — keep conversion below 10–15% over the assay window to maintain initial-rate conditions. Titrate inhibitor across at least 8–10 concentrations spanning 3 logs around the expected IC50.
Can I use recombinant MMP-25 as a positive control for Western blot with the RP-MMP25 rabbit polyclonal antibody?
Yes — this is one of the primary use cases for pairing REC-MMP25 with antibody RP-MMP25. Because both are produced in-house at Triple Point Biologics, the recombinant and antibody are guaranteed compatible for Western blot validation. Load 20–50 ng of REC-MMP25 per lane on a standard 10–12% SDS-PAGE gel. Run alongside your cell lysate samples. RP-MMP25 should detect the recombinant band at 65–72 kDa, providing an unambiguous size marker and confirming antibody performance in the same experiment.
How much recombinant MMP-25 should I load for a Western blot positive control lane and what antibody dilution works with RP-MMP25?
Load 20–50 ng of REC-MMP25 per lane for a clear, non-saturating positive control signal. For antibody RP-MMP25, a primary dilution of 1:500–1:2000 in 5% non-fat milk or BSA in TBST is a standard starting range for Western blot; optimize for your detection system (ECL vs. fluorescent secondary). The HEK293-expressed recombinant retains the mammalian epitope context, minimizing false-negative results that can occur with bacterially expressed antigens. RP-MMP25 product page (/anti-mmp-25-rabbit-polyclonal-antibody) lists validated dilution ranges and blocking conditions.
How should I store recombinant MMP-25 and what is the shelf life after thawing?
Store REC-MMP25 at -20°C in single-use aliquots. Repeated freeze-thaw cycles degrade metalloproteinase activity — even two cycles can produce measurable losses in specific activity. Once thawed, keep on ice and use within the same working session; do not re-freeze. Under recommended storage conditions, activity is stable for 12 months from receipt. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol) is formulated to stabilize the zinc-metalloproteinase fold; do not dialyze away glycerol unless immediately proceeding to assay.
Validation imagery coming soon
Western blot validation figures for REC-MMP25 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.