Recombinant Mouse Spinesin Protein, CF

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

Recombinant Mouse 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 >1,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Spinesin protein
  Tyr61-Arg445, with an N-terminal 7-His tag
  Accession # NP_109634
• Accession #: NP_109634
• 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: 55-61 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, pH 7.5
  • Assay Buffer: 100 mM Tris, pH 8.5
  • Recombinant Mouse Spinesin (rmSpinesin) (Catalog # 1928-SE)
  • Trypsin (Sigma, Catalog # T-1426)
  • Recombinant Human Serpin A1/ alpha 1‑Antitrypsin (rhSerpin A1) (Catalog # 1268-PI)
  • Fluorogenic Peptide Substrate: BOC-Gln-Ala-Arg-AMC (Catalog # ES014)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmSpinesin to 200 µg/mL in Activation Buffer.
  2. Dilute Trypsin to 0.4 µg/mL in Activation Buffer.
  3. Combine equal volumes of 200 µg/mL rmSpinesin and 0.4 µg/mL Trypsin. Include a Trypsin control containing Activation Buffer in place of rmSpinesin.
  4. Incubate at room temperature for 1 hour.
  5. Dilute rhSerpin A1 to 36 µg/mL in Assay Buffer.
  6. Dilute the reaction mixtures in half by adding rhSerpin A1 for final concentrations of 18 µg/mL rhSerpin A1, 50 µg/mL rmSpinesin, and 0.1 µg/mL Trypsin. Addition of rhSerpin A1 will stop the activity of Trypsin. Control from step 3 will verify that rhSerpin A1 is working properly.
  7. Dilute rmSpinesin to 2 ng/µL in Assay Buffer. Dilute Trypsin control equally.
  8. Dilute Substrate to 200 µM in Assay Buffer.
  9. Load 50 µL of the diluted samples into a black well plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
  10. Read at excitation and emission wavelengths of 380 nm and 460 nm, 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 (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • rmSpinesin: 0.1 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 Mouse 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 new member of type II transmembrane serine proteases (TTSPs) (1). Mouse Spinesin contains the following structural domains: a short N-terminal cytoplasmic tail, a transmembrane domain, a stem region and a serine protease domain (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. There could be 4 types of transcripts due to alternative splicing (3). Type 4 predicts 10 extra amino acids at the N-terminus as compared to type 3. The ectodomain corresponding to type 3 (residues 61‑445) or  type 4 (residues 71‑455) was expressed and purified as a single chain pro-enzyme. By SDS‑PAGE, the pro‑enzyme migrates as multiple forms, possibly due to differential glycosylation. The pro‑enzyme can be activated by trypsin treatment. The resulting enzyme is active and its activity is measured as described above. The activated enzyme is a disulfide bond‑linked dimer.

1. Shibata, K. et al. (2000) Genome Res. 10:1757.
2. Yamaguchi, Y. et al. (2002) J. Biol. Chem. 277:6806.
3. Watanable, Y. et al. (2004) Biochem. Biophys. Res. Commun. 324:333.

Bioinformatics

• Gene Symbol: Tmprss5
• Uniprot:
  • NP_109634()