Recombinant Mouse Tryptase epsilon/BSSP-4 Protein, CF

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

Recombinant Mouse Tryptase epsilon/BSSP-4 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 >5,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Tryptase epsilon/BSSP-4 protein
  Ala33-Ser306, with a C-terminal 10-His tag
• Accession #: Q7TML0
• N-terminal Sequence: Ala33
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Prss22
• 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: 37 kDa and 33 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 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 and 150 mM NaCl, pH 7.5.
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, pH 7.5 (TCN)
  • Assay Buffer: 50 mM Tris, 0.25 M NaCl, pH 8.0
  • Recombinant Mouse Tryptase-epsilon /BSSP-4 (rmBSSP-4) (Catalog # 2059-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 in DMSO
  • 5,5'Dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma Catalog # D-8130), 10 mM in DMSO
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Reconstitute or dilute rmBSSP-4 to 200 µg/mL with Activation Buffer.
  2. Dilute Thermolysin to 0.4 µg/mL with Activation Buffer.
  3. Mix equal volumes of 200 µg/mL rmBSSP-4 and 0.4 µg/mL Thermolysin for final concentrations of 100 µg/mL and 0.2 µg/mL, respectively.
  4. Incubate at 37 °C for 30 minutes.
  5. Stop the reaction with 10 mM 1,10 Phenanthroline.
  6. Dilute activated rmBSSP to 0.4 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 0.4 ng/µL rmBSSP into 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 rmBSSP.
  9. Read at absorbance wavelength 405 nm 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:
  • rmBSSP-4: 0.020 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 epsilon/BSSP-4 Protein, CF

• BSP-2
• BSSP4
• BSSP-4
• hBSSP-4
• MGC9599
• prosemin
• protease, serine S1 family member 22
• protease, serine, 22
• PRSS22
• serine protease 22
• serine protease 26
• SP001LA
• Tryptase epsilon
• UNQ302/PRO343

Background

Tryptase epsilon , brain specific serine protease 4 (BSSP-4) and brain serine protease 2 (BSP-2) are different names given for the same serine protease that is encoded by the PRSS22 gene (1-3). Initially identified having brain-specific expression, mouse Tryptase epsilon is preferentially expressed in epithelium‑rich tissues such as the lung and eye, which is similar to its human counterpart (3). The mouse protein is synthesized with a signal peptide (amino acid residues 1 to 32), a pro peptide (residues 33 to 49) and a mature chain (residues 50 to 306) corresponding to the serine protease domain. The full-length protein was expressed and the secreted protein purified. The N-terminal sequencing result indicates that the purified protein corresponds to the pro enzyme. After activation with thermolysin, the enzyme has low activity against peptide substrates tested, but high activity against thioester substrates. The thioester activity is inhibited by 2 mM AEBSF (Catalog # EI001), a general serine protease inhibitor, and by recombinant human Serpin A5 (Catalog # 1266-PI).

1. Wong, G.W. et al. (2001) J. Biol. Chem. 276:49169.
2. Davies, B.J. et al. (1998) J. Biol. Chem. 273:23004.
3. Wong, G.W. et al. (2004) J. Biol. Chem. 279:2438.

Bioinformatics

• Gene Symbol: Prss22
• Uniprot:
  • Q7TML0()