TLL-1 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-TLL1
In stock
- SKU
- REC-TLL1
Target Overview
TLL-1 (Tolloid-like protein 1; UniProt O43897) is a secreted, zinc-dependent metalloprotease of the astacin/BMP-1 family encoded by the TLL1 gene on human chromosome 4q32. The full-length human protein spans 1,013 amino acids. This recombinant form is produced in HEK293 mammalian cells, a system that supports the post-translational processing — including signal peptide cleavage, glycosylation, and pro-domain removal — expected of a secreted extracellular protease, making the resulting protein well-suited for enzymatic characterisation under near-physiological conditions. In the extracellular matrix, TLL-1 cleaves the C-propeptides of fibrillar procollagens and processes additional substrates including chordin, pro-biglycan, and pro-lysyl oxidase. These activities position it as a key regulator of BMP signalling gradients and connective tissue assembly. Recombinant TLL-1 is used in substrate cleavage assays to define specificity and kinetics, and in inhibitor screens to identify small molecules or endogenous regulators that modulate its activity. Because chordin cleavage by BMP-1/tolloid family proteases is well characterised biochemically, recombinant TLL-1 serves as a direct comparator in selectivity studies within this enzyme family. Researchers performing antibody validation — confirming that an anti-TLL1 antibody detects the correct band by Western blot or the correct tissue localisation by IHC — can use this recombinant as a defined positive control antigen. It pairs directly with the Triple Point Biologics matched antibody (SKU: RP-TLL1), which targets the same human TLL-1 protein. The HEK293 expression system produces a glycosylated, secreted protein consistent with the endogenous form found in human biological samples, supporting direct comparability in validation experiments.
Background
Applications
- Chordin cleavage activity assay (SDS-PAGE or ELISA-based detection of cleavage fragments)
- Pro-biglycan and pro-lysyl oxidase substrate processing assay
- SLIT2 cleavage and axon guidance substrate characterisation in vitro
- Small-molecule inhibitor IC50 determination against recombinant TLL-1 protease activity
- Antibody validation positive control for Western blot using matched antibody RP-TLL1
- Enzyme kinetics (Km, kcat) characterisation with synthetic or recombinant substrates
- Disease-variant functional comparison: side-by-side activity assay of wild-type vs. gain-of-function TLL-1 constructs
- Binding and biophysical characterisation (SPR, BLI) of TLL-1 interactions with endogenous inhibitors or substrates
References
- Jones LE et al. SLIT2 repellent is cleaved by TLL1 protease and promotes sensory axon fasciculation. Development. 2026. doi:10.1242/dev.205124. PMID: 41626796.
- Hao JL et al. TLL1 knockdown attenuates prostate cancer progression by enhancing antitumor immunity. Oncogene. 2025. doi:10.1038/s41388-025-03517-7. PMID: 40760092.
- Shukla AK. Computational simulations aided prioritization of genomic targets for congenital heart disease (CHD) against developmental toxicity. Reprod Toxicol. 2025. doi:10.1016/j.reprotox.2025.108940. PMID: 40334872.
- Lee J et al. ETV5 reduces androgen receptor expression and induces neural stem-like properties during neuroendocrine prostate cancer development. Proc Natl Acad Sci U S A. 2025. doi:10.1073/pnas.2420313122. PMID: 40117308.
- Agam N et al. Mitral Valve Prolapse Caused by TLL1 Gain-of-Function Mutation. Can J Cardiol. 2025. doi:10.1016/j.cjca.2025.01.018. PMID: 39880331.
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 recombinant TLL-1 run at on SDS-PAGE or Western blot?
The full-length human TLL-1 precursor is 1,013 amino acids, giving a predicted unprocessed molecular weight of ~116 kDa. Because this recombinant is produced in HEK293 cells, it undergoes signal peptide cleavage and N-linked glycosylation, so the mature, secreted form typically resolves between 120–130 kDa under denaturing, reducing conditions — somewhat above the sequence-predicted value due to glycan addition. If you observe a lower-molecular-weight species (~85–95 kDa), this likely reflects pro-domain removal. Run alongside a broad-range molecular-weight ladder and use anti-TLL-1 antibody RP-TLL1 for confirmation.
Which processing form of TLL-1 does this recombinant represent — zymogen or active enzyme?
REC-TLL1 is supplied as the active enzyme form. HEK293 expression supports the endogenous-like secretory processing of TLL-1, including pro-domain removal that occurs either autocatalytically or via furin-type proprotein convertases during transit through the secretory pathway. The resulting protein retains the catalytic astacin domain, the CUB domains, and the EGF-like domain, which together constitute the functional extracellular form. If your experiment requires the zymogen, the pro-domain would need to be re-introduced separately; REC-TLL1 is not suited for that purpose as supplied.
What substrates can I use to measure TLL-1 protease activity in a cleavage assay?
TLL-1 cleaves the C-propeptides of fibrillar procollagens (particularly procollagen I and II), chordin, pro-biglycan, and pro-lysyl oxidase. For biochemical assays, fluorogenic peptide substrates derived from the procollagen C-propeptide cleavage site or the chordin BMP-binding domains work well. Recombinant chordin is the most commonly used macromolecular substrate for activity confirmation, with cleavage products (~65 kDa and ~45 kDa) visible by SDS-PAGE and Western blot. For kinetic measurements, fluorescence-quenched peptide mimetics of the scissile bond region allow continuous-read assay formats compatible with standard plate readers.
What buffer conditions should I use for TLL-1 activity assays and is calcium required?
REC-TLL1 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. Calcium is essential for astacin-family metalloprotease activity and must be maintained in your assay buffer; a working concentration of 2–5 mM CaCl2 is recommended. Reactions are typically run at pH 7.2–7.5 at 37 °C. Avoid chelating agents such as EDTA or EGTA, which will rapidly inactivate the enzyme. Zinc-chelating inhibitors (e.g., 1,10-phenanthroline) serve as useful negative controls. Detergents above 0.01% Tween-20 may reduce activity and should be tested empirically.
What starting concentration of recombinant TLL-1 should I use in an inhibitor screen or IC50 assay?
A starting enzyme concentration of 1–5 nM is appropriate for most fluorogenic substrate IC50 assays, keeping enzyme well below substrate Km to satisfy the assumptions of standard inhibitor kinetics. Confirm that substrate conversion remains linear (typically <20% depletion) over your measurement window before running compound titrations. For pull-down or SPR binding experiments, 50–200 nM is a practical loading range. Because REC-TLL1 is supplied at >90% purity by SDS-PAGE, active-site titration with a tight-binding reference inhibitor (e.g., actinonin or a hydroxamate tool compound) is advisable to establish the active fraction before calculating Ki or IC50 values.
Can I use REC-TLL1 as a positive control for Western blot with the matched anti-TLL-1 antibody RP-TLL1?
Yes — this is the primary intended use of the pairing. REC-TLL1 and RP-TLL1 are produced and validated in the same laboratory, so band confirmation is guaranteed. Load 10–50 ng of REC-TLL1 per lane; at 20 ng you should see a clean band at 120–130 kDa with RP-TLL1 at a 1:1,000–1:2,000 dilution using standard HRP-secondary/ECL detection. This positive-control lane is especially useful when probing low-expressing tissue lysates or verifying antibody lot-to-lot consistency. RP-TLL1 details and protocol notes are at /anti-tll-1-rabbit-polyclonal-antibody.
How much recombinant TLL-1 should I load for Western blot antibody validation with RP-TLL1?
For antibody validation purposes, a two- or three-point dilution series — 10 ng, 25 ng, and 50 ng per lane — establishes linearity and helps set exposure parameters alongside your experimental lysate lanes. At 25 ng, REC-TLL1 produces a robust signal at 120–130 kDa with RP-TLL1 at 1:1,000 dilution, well within the linear dynamic range of standard film or digital imaging systems. Including this titration in IHC validation panels also provides a reliable mass anchor when comparing tissue staining intensity across antibody lots.
How should I store and handle REC-TLL1 to maintain enzyme activity, and what is its shelf life?
REC-TLL1 is shipped on dry ice and should be stored at -20 °C immediately upon receipt in single-use aliquots. Repeated freeze-thaw cycles measurably reduce metalloprotease activity — plan aliquot sizes around your typical single-experiment usage (e.g., 5–10 µg per tube). Shelf life is 12 months from the manufacture date when stored correctly. Working dilutions should be prepared fresh in assay buffer containing 2–5 mM CaCl2, kept on ice, and used within 4 hours. Do not dilute into buffers containing EDTA, EGTA, or reducing agents such as DTT above 0.5 mM, as these will chelate the active-site zinc.
Validation imagery coming soon
Western blot validation figures for REC-TLL1 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.