Leishmanolysin (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-Leishmanolysin
In stock
- SKU
- REC-Leishmanolysin
Target Overview
Leishmanolysin-like peptidase (LMLN), also known as Invadolysin, is a 647-amino-acid zinc-dependent metalloprotease encoded by the LMLN gene (UniProt Q96KR4). It belongs to the M8 family of metalloproteases — a family named for the Leishmania surface protease leishmanolysin (GP63) — and retains the conserved HEXXH zinc-binding motif characteristic of that lineage. LMLN localises to the cytoplasm and has been characterised in the context of cell migration, mitotic regulation, and intracellular proteolytic signalling. This recombinant form is expressed in HEK293 cells, providing mammalian post-translational processing including glycosylation patterns relevant to human biology. The full-length sequence spans residues 1–647. HEK293 expression is particularly suited to this target because correct folding of the metalloprotease domain and disulfide architecture are difficult to achieve in prokaryotic systems. Researchers use this recombinant for several bench applications: (1) metalloprotease substrate profiling, in which candidate peptide or protein substrates are incubated with the enzyme and cleavage monitored by fluorogenic assay or LC-MS/MS; (2) small-molecule inhibitor screens, using FRET-based or gel-shift readouts to determine IC50 values; (3) antibody validation, as a well-characterised positive control antigen for Western blot and immunohistochemistry — researchers running LMLN detection panels can pair this recombinant directly with the matched Triple Point Biologics polyclonal antibody (RP-Leishmanolysin); and (4) structural and biophysical characterisation, including thermal shift assays and SPR binding studies with candidate interactors. The recombinant is supplied as research-use-only material and is not intended for diagnostic or therapeutic application.
Background
Applications
- Metalloprotease activity assay using fluorogenic peptide substrates (e.g., FRET-quenched peptides bearing the HEXXH-site cleavage motif)
- Small-molecule inhibitor IC50 determination by gel-shift or fluorescence-based kinetic assay
- Substrate identification by incubation with candidate proteins followed by LC-MS/MS cleavage-site mapping
- Antibody validation positive control for Western blot — paired use with Triple Point Biologics RP-Leishmanolysin polyclonal
- Antibody validation positive control for IHC tissue section staining, using recombinant as spike-in or dot-blot standard
- Thermal shift (differential scanning fluorimetry) assay to characterise ligand and inhibitor binding to the metalloprotease domain
- SPR or bio-layer interferometry binding kinetics with candidate interacting proteins or metalloprotease inhibitor scaffolds
- Recombinant antigen for ELISA standard curve construction in quantitative LMLN detection assays
References
Additional Specifications
| Storage Buffer | 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol |
|---|---|
| Endotoxin Level | <0.1 EU/µg by LAL |
| Purity (%) | >90% by SDS-PAGE |
| Expression System | HEK293 |
| Subcellular Localization | Subcellular localization not yet annotated |
Frequently Asked Questions
What molecular weight should I expect for recombinant Leishmanolysin (LMLN) on SDS-PAGE?
The full-length recombinant LMLN (residues 1–647) has a predicted molecular weight of approximately 73 kDa from sequence alone. However, because this protein is expressed in HEK293 cells, N-linked glycosylation typically shifts the apparent molecular weight on SDS-PAGE to ~80–90 kDa. If you observe a band in that range under reducing conditions, that is expected behavior — not degradation. Running a non-reducing lane alongside can help distinguish glycoforms from disulfide-linked dimers. >90% purity by SDS-PAGE is confirmed for each lot.
What isoform or processing state is the recombinant LMLN protein — full-length, pro-form, or mature?
This recombinant corresponds to the full-length human LMLN sequence, residues 1–647 (UniProt Q96KR4), expressed in HEK293 cells. It is supplied as an active enzyme rather than a zymogen pro-form. The conserved HEXXH zinc-binding motif of the M8 metalloprotease family is intact. No propeptide truncation has been introduced. If your experiment requires a defined mature-domain fragment, we recommend verifying autoproteolytic processing status by comparing freshly thawed aliquot bands against the expected ~80–90 kDa full-length position before committing to activity assays.
What substrates does recombinant LMLN (Leishmanolysin-like peptidase) cleave in vitro?
LMLN is a zinc-dependent metalloprotease of the M8 family with documented activity against extracellular matrix components and intracellular signalling substrates linked to mitotic regulation and cell migration. In vitro, fluorogenic peptide substrates containing the general metalloprotease consensus cleavage sequence (e.g., Mca-RPPGFSAFK(Dnp)-NH₂ or similar FRET-quenched peptides used for M8 family members) can be used to monitor activity. Substrate Km and kcat values should be empirically determined for your specific peptide series; published GP63 family substrate preferences provide a reasonable starting framework.
What activity assay buffer should I use for recombinant Leishmanolysin (LMLN) metalloprotease activity assays?
The protein is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂. For activity assays, this storage buffer is a suitable starting point. Calcium (5 mM) is required to maintain metalloprotease domain stability. Avoid EDTA or EGTA in assay buffers, as chelation of Zn²⁺ or Ca²⁺ will abolish activity. If diluting for kinetic assays, maintain at least 1 mM CaCl₂ in the working buffer. Zinc-dependent activity can be confirmed using a 10 µM ZnCl₂ supplementation control and ablated with 10 mM 1,10-phenanthroline as a positive inhibitor control.
What is a good starting concentration of recombinant LMLN for an in vitro metalloprotease activity assay?
A practical starting point is 0.1–1.0 µg/mL of recombinant LMLN per reaction, depending on substrate concentration and fluorescence sensitivity of your plate reader. At 1 µM fluorogenic peptide substrate, 0.5 µg/mL enzyme typically yields a linear signal window over 30–60 minutes at 37°C. Titrate enzyme concentration to confirm linearity before running inhibitor IC₅₀ curves. Note that glycerol carried over from the storage buffer (10%) can mildly suppress activity at high protein loads, so dilute at least 1:5 into assay buffer before use.
Can I use recombinant Leishmanolysin (LMLN) as a Western blot positive control with the RP-Leishmanolysin antibody?
Yes — REC-Leishmanolysin and RP-Leishmanolysin (the matched rabbit polyclonal, available at /anti-leishmanolysin-rabbit-polyclonal-antibody) are developed in the same program specifically for this pairing. The antibody is validated for Western blot, and recombinant protein loaded alongside lysate provides a defined-size positive control at the expected ~80–90 kDa glycosylated band. This is particularly useful when validating the antibody against novel cell lines or tissue lysates where endogenous LMLN expression level is uncertain.
How much recombinant Leishmanolysin should I load for a Western blot positive control lane?
Load 20–50 ng of REC-Leishmanolysin per lane as a positive control alongside your cell lysate samples. At 50 ng under reducing SDS-PAGE conditions, RP-Leishmanolysin (matched rabbit polyclonal, SKU RP-Leishmanolysin) reliably detects the ~80–90 kDa band at antibody dilutions of 1:1,000–1:2,000. If your membrane has high background, dropping to 20 ng per lane minimizes the risk of signal saturation in the recombinant lane while still providing a clear reference point for band position.
How should I store and handle recombinant Leishmanolysin (LMLN) to maintain activity over time?
Store at -20°C in the single-use aliquots supplied. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂) stabilizes the metalloprotease domain during freeze-thaw. Repeated freeze-thaw cycles degrade zinc-metalloprotease activity measurably — typically >20% loss per additional cycle. On thaw, keep on ice and use within the same working session. Do not dilute working stocks more than 24 hours in advance, as dilute metalloprotease solutions below ~10 µg/mL are prone to adsorptive losses on polypropylene tubes; add 0.1% BSA (protease-free) to diluted working stocks if same-day use is not possible.
Validation imagery coming soon
Western blot validation figures for REC-Leishmanolysin 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.