Recombinant Human Spinesin Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Spinesin Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate Boc-QAR-AMC (Catalog # ES014). The specific activity is >300 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Spinesin protein
  Tyr71-Leu457 (Phe369Leu), with an N-terminal 9-His tag
  Accession # NP_110397
• Accession #: NP_110397
• N-terminal Sequence: His
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TMPRSS5
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 43 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.
• SDS-PAGE: 63 kDa, 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 MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, pH 8.0
  • Recombinant Human Spinesin (rhSpinesin) (Catalog # 2495-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • Substrate: BOC-Gln-Ala-Arg-AMC (Catalog # ES014), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhSpinesin to 200 µg/mL in Activation Buffer.
  2. Dilute Thermolysin to 2 µg/mL in Activation Buffer.
  3. Combine equal amounts of 200 µg/mL rhSpinesin and 2 µg/mL Thermolysin.
  4. Incubate the reactions at 37 °C for 1 hour.
  5. Stop the Thermolysin activity by adding 1,10 Phenanthroline to a final concentration of 10 mM.
  6. Dilute activated rhSpinesin to 2 ng/µL in Assay Buffer.
  7. Dilute Substrate to 200 µM in Assay Buffer.
  8. Load 50 µL of 2 ng/µL rhSpinesin into a plate, and start the reaction by adding 50 µL of 200 µM Substrate.
  9. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
  10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  11. 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 (Sigma, Catalog # A-9891).

  Per Well:
  • rhSpinesin: 0.100 µg
  • Thermolysin: 0.001 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Spinesin Protein, CF

• EC 3.4.21
• EC 3.4.21.-
• EC 3.4.21.4
• MGC141886
• MGC148044
• Spinesin
• TMPRSS5
• transmembrane protease serine 5
• transmembrane protease, serine 5

Background

Spinesin, encoded by the TMPRSS5 gene, is a member of type II transmembrane serine proteases (TTSPs) (1). Human Spinesin contains the following structural domains: a short N-terminal cytoplasmic tail (amino acid residues 1-49), a transmembrane domain (residues 50-70), a stem region and a serine protease domain (residues 71-457) (2). The domain structure of Spinesin is common to other TTSPs, many of which have additional domains. The stem region of Spinesin contains a scavenger receptor-like domain. The ectodomain of human Spinesin (residues 71-457) was expressed and purified as a single chain pro-enzyme. The deduced amino acid sequence contains a Leu instead of a Phe residue at position 369; the former is identical to the mouse protein (3, 4). The pro-enzyme can be activated and the resulting enzyme activity can be measured as described in the Activity Assay Protocol.

1. Hooper, J.D. et al. (2001) J. Biol. Chem. 276:857.
2. Yamaguchi, Y. et al. (2002) J. Biol. Chem. 277:6806.
3. Carninci, P. et al. (2000) Genome Res. 10:1617.
4. Shibata, K. et al. (2000) Genome Res. 10:1757.

Publications for Spinesin (2495-SE)(1)

Publication using 2495-SE

• Buzza M, Martin E, Driesbaugh K, Desilets A, Leduc R, Antalis T Prostasin is required for matriptase activation in intestinal epithelial cells to regulate closure of the paracellular pathway. J Biol Chem, 2013-02-26;288(15):10328-37. 2013-02-26 [PMID: 23443662] (Bioassay, Human)

Bioinformatics

• Gene Symbol: TMPRSS5
• Uniprot:
  • NP_110397()