Measured by its ability to cleave a fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (Catalog # ES003). The specific activity is >500 pmol/min/µg, as measured under the described conditions.
Note:It is extremely important that the assay solution does not contain salt as it inhibits TACE activity.
Mouse myeloma cell line, NS0-derived mouse TACE/ADAM17 protein Pro18-Asp563 and Arg215-Asp563, both with a C-terminal 10-His tag, Pro18-Arg214 and Asp59-Arg214
Recombinant Mouse TACE/ADAM17 is prone to proteolytic cleavage at C-terminus.
tag may not be present in the preparation.
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
<1.0 EU per 1 μg of the protein by the LAL method.
63 kDa, 40 kDa, 23 kDa, 18 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Substrate: MCA-Pro-Leu-Ala-Gln-Ala-Val-DPA-Arg-Ser-Ser-Ser-Arg-NH2 (Catalog # ES003), 2 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rmTACE to 0.2 ng/µL in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
In a plate, load 50 µL of 0.2 ng/µL rmTACE to wells, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank of 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)
*Adjusted for Substrate Blank **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
rmTACE: 0.01 µg
Substrate: 10 µM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Mouse TACE/ADAM17 Protein, CF
ADAM metallopeptidase domain 17
ADAM metallopeptidase domain 18
disintegrin and metalloproteinase domain-containing protein 17
Snake venom-like protease
TNF-alpha converting enzyme
tumor necrosis factor, alpha, converting enzyme
Tumor necrosis factor alpha (TNF-alpha ) converting enzyme (TACE) is a member of the ADAM family that contains a disintegrin and metalloprotease-like domain. The domain structure of mouse TACE consists of pro (aa 18-214), catalytic (aa 215-474), disintegrin-like (aa 475-563), cysteine-rich (aa 564-602), Crambin-like (aa 603-671), transmembrane (aa 672-692), and cytoplasmic (aa 693-827) (1, 2). Recombinant mouse TACE ends after the disintegrin-like domain and consists of several forms as described. In addition to its ability to release the 17 kDa extracellular form of TNF-alpha from the 26 kDa membrane-anchored TNF-alpha, TACE also plays an essential role in shedding ectodomains from a variety of proteins such as L-selectin, transforming growth factor-alpha, amyloid protein precursor, and Notch1 receptor. TACE mRNA is present in virtually every tissue and TACE protein resides both on the cell surface and in the cell (3).
Cerretti, D.P. et al. (1999) Cytokine 11:541.
Mizui, Y. et al. (1999) Gene 233:67.
Black, R.A. (2004) Handbook of Proteolytic Enzymes, Barrett, A.J. et al.eds. pp. 726, Academic Press, San Diego.
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