Recombinant Active Human Hepsin His-tag Protein, CF Summary
Details of Functionality |
Measured by its ability to cleave tert-butoxycarbonyl-Gln-Arg-Arg-7-amino-4-methylcoumarin (Boc-QRR-AMC). The specific activity is >45,000 pmol/min/μg, as measured under the described conditions. |
Source |
Mouse myeloma cell line, NS0-derived human Hepsin protein Arg45 to Leu417 (Asp161Glu, Arg162Lys) with a C-terminal 10-His tag The proform was activated. |
Accession # |
|
N-terminal Sequence |
Arg45 (non-catalytic chain) & Ile163 (catalytic chain) |
Structure / Form |
Active form |
Protein/Peptide Type |
Recombinant Enzymes |
Purity |
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Endotoxin Note |
<0.10 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
Dilutions |
|
Theoretical MW |
13 kDa (non-catalytic chain), 29 kDa (catalytic chain). 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. |
SDS-PAGE |
17-24 and 29-33 kDa, under reducing conditions |
Packaging, Storage & Formulations
Storage |
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.- 6 months from date of receipt, -20 to -70 °C as supplied.
- 3 months, -20 to -70 °C under sterile conditions after opening.
|
Buffer |
Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
Purity |
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Assay Procedure |
- Assay Buffer: 50 mM Tris, 0.05% Brij-35, pH 9.0
- Recombinant Active Human HepsinHis-tag (rhHepsin) (Catalog # 11416-SE)
- Substrate: Boc-QRR-AMC, 5 mM stock in DMSO
- Black 96-well Plate
- Plate Reader with Fluorescence Read Capability
- Dilute rhHepsin, active to 0.01 µg/mL in Assay Buffer.
- Dilute Substrate to 400 µM in Assay Buffer.
- Load in a plate 50 μL of 0.01 µg/mL rhHepsin, active, and start the reaction by adding 50 μL of 400 μM Substrate. Include a Substrate Blank containing 50 μL of 400 μM Substrate and 50 μL of Assay Buffer.
- Read at excitation and emission wavelengths of 380 nm and 460 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 7-amino, 4-Methyl Coumarin Per Well - rhHepsin, active: 0.0005 μg
- Substrate: 200 µM
|
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Active Human Hepsin His-tag Protein, CF
Background
Hepsin, also known as TMPRSS1, is part of a family of type II transmembrane serine proteases (TTSPs) (1) that cleaves critical target proteins including Hepatocyte growth factor (HGF), uromodulin (UMOD), uPA, and macrophage-stimulating protein (MSP) at specific cleavage sites following basic residues with a strong preference for arginine residues (2-5). Hepsin is a 417 amino acid polypeptide that is activated to a two-chain disulfide-linked form. Hepsin, like other proteases in this family, contains an N-terminal cytoplasmic tail and transmembrane domain, and an extracellular stem region with an SRCR domain and C-terminal serine protease domain with a conserved activation site (1, 6). It is most highly expressed in liver, but is also present in many other tissues, notably lung, kidney, and skeletal muscle (7) and is found at high levels in several cancers including prostate, breast, and ovarian (2, 5, 8-10). In the liver, hepsin functions to regulate glucose, lipid, and protein metabolism through proteolytic conversion of pro-HGF to HGF, a potent ligand for Met signaling and dysregulation (11). Involvement in cancers has made hepsin a target of interest using either small molecule inhibitors or antibodies to neutralize their proteolytic activity, for example for prostate cancer and metastasis (12-14). Hepsin is also of interest for its potential role in osteoarthritis (15) and its ability to cleave surface proteins of respiratory viruses making it a therapeutic target for respiratory viral diseases (13). Recombinant Human Hepsin was expressed as a secreted, soluble protein lacking its cytosolic and transmembrane domains and activated.
- Li. S. et al. (2021) FEBS J. 288:5252.
- Herter, S. et al. (2005) Biochem. J. 390:125.
- Ganesan, R. et al. (2011) Mol. Cancer Res. 9:1175.
- Brunati, M. et al. (2015) Elife 4:e08887.
- Tanabe, L.M. and K. List (2017) FEBS J. 284:1421.
- Somoza, J.R. et al. (2003) Structure 11:1123.
- Tsuji, A. et al. (1991) J. Biol. Chem. 266:16948.
- Dhanasekaran, S.M. et al. (2001) Nature 412:822.
- Miao, J. et al. (2008) Int. J. Cancer 123:2041.
- Xing, P. et. al. (2011) J. Investig. Med. 59:803.
- Li, S. et al. (2020) Proc. Natl. Acad. Sci. USA 117:12359.
- Damalanka, V.C. et al. (2019) J. Med. Chem. 62:480.
- Murza, A. et al. (2020) Expert Opin. Ther. Pat. 30:807.
- Wu, Q. and Parry, G. (2007) Front. Biosci. 12:5052.
- Wilkinson, D.J. et al. (2017) Sci. Rep. 7:16693.
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