Recombinant Mouse Tryptase-5/Prss32 Protein, CF

• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Tryptase-5/Prss32 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Arg-ThioBenzyl ester (Z-R-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >400 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Tryptase-5/Prss32 protein
  Ser20-Arg304, with a C-terminal 10-His tag
• Accession #: NP_081496
• N-terminal Sequence: Ser20
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Prss32
• 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: 32 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: 40 kDa doublet, 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 200 μg/mL in sterile 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl and 0.05% Brij-35 pH 7.5.
• Assay Procedure:
  • Activation Buffer: (TCNB) 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5
  • Assay Buffer: 50 mM Tris, 0.25 M NaCl, pH 8.0
  • Recombinant Mouse Tryptase‑5/Prss32 (rmTPS-5) (Catalog # 2634-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • Substrate: Z-Arg-SBzl (SM Biochemicals, Catalog # SMSB01), 10 mM stock in DMSO
  • 5,5’Dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma Catalog # D-8130)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmTPS-5 to 100 µg/mL with Activation Buffer.
  2. Dilute Thermolysin to 0.4 µg/mL with Activation Buffer.
  3. Mix equal volumes of 100 µg/mL rmTPS-5 and 0.4 µg/mL Thermolysin for final concentrations of 50 µg/mL and 0.2 µg/mL, respectively.
  4. Incubate at 37 °C for 1 hour.
  5. Stop the reaction with 10 mM 1,10 Phenanthroline.
  6. Dilute activated rmTPS-5 to 4 ng/µL in Assay Buffer.
  7. Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
  8. Load 50 µL of the 4 ng/µL rmTPS-5 in a clear plate and start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate mix without any rmTPS-5.
  9. Read at 405 nm (Absorbance), in kinetic mode for 5 minutes.
  10. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 13260 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rmTPS-5: 0.20 µg
  • DTNB: 100 µM
  • 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 Tryptase-5/Prss32 Protein, CF

• EOS
• Prss32
• Tryptase5
• Tryptase-5

Background

Tryptase-5 is encoded by Prss32, one of 13 genes on mouse chromosome 17A3.3 that correspond to functional trypsin-like serine proteases (1). The deduced amino acid sequence of mouse Tryptase-5 consists of 331 residues with a signal peptide (residues 1 to 19), a pro region (residue 20 to 53), a catalytic domain (54 to 304), and a C-terminal hydrophobic peptide (residues 305 to 331). The mRNA is expressed in smooth muscle, eye, stomach, uterus and lymph node. Apparently, Prss32 does not seem to have a counterpart in the human genome. The mouse Tryptase-5 (residues 20 to 304) was expressed in the NS0 cells with a foreign signal peptide. The protein was secreted and purified. After being treated by thermolysin, the enzyme is active against a peptide substrate as described in the Activity Assay Protocol.

1. Wong, G. W. et al. (2004) J. Biol. Chem. 279:2438.

Bioinformatics

• Gene Symbol: Prss32
• Uniprot:
  • NP_081496()