Recombinant Human Tryptase alpha/beta-1 Protein, CF

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

Recombinant Human Tryptase alpha/beta-1 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >650 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Tryptase alpha/beta-1 protein
  Met1-Pro275, with a C-terminal 10-His tag
• Accession #: Q15661.1
• N-terminal Sequence: Val29
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TPSB2
• 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: 29 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: 38 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Maturation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Heparin Incubation Buffer (HIB): 100 µg/mL Heparin (Sigma, Catalog # H3393), 50 mM MES, pH 5.5
  • Assay Buffer: 50 mM Tris, pH 8.5
  • Recombinant Human Tryptase alpha/beta-1 (rhTPSAB1) (Catalog # 3796-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 2 mM stock in DMSO
  • 1, 10 Phenanthroline (Sigma, Catalog # 320056)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhTPSAB1 to 100 µg/mL in Maturation Buffer containing Thermolysin at a final concentration of 0.1 µg/mL.
  2. Incubate at room temperature for 15 minutes.
  3. Stop Thermolysin activity by adding an equal volume of 10 mM 1, 10 Phenanthroline prepared in HIB.
  4. Dilute the matured rhTPSAB1 to 10 µg/mL in HIB.
  5. Incubate for 2 hours at room temperature.
  6. Dilute 10 µg/mL rhTPSAB1 to 2.5 µg/mL in HIB.
  7. Dilute 2.5 µg/mL rhTPSAB1 to 0.50 µg/mL in Assay Buffer.
  8. Dilute Substrate to 20 µM in Assay Buffer.
  9. Load 50 µL of 0.50 µg/mL rhTPSAB1 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
  10. Read at excitation and emission wavelengths of 320 nm and 405 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhTPSAB1: 0.025 µg
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Human Tryptase alpha/beta-1 Protein, CF

• EC 3.4.21
• EC 3.4.21.59
• mast cell tryptase beta II
• mast cell tryptase beta III
• TPS2
• TPS2tryptase beta-2
• TPSB2
• Tryptase alpha/beta-1
• tryptase beta 2 (gene/pseudogene)
• tryptase beta 2
• tryptase beta II
• tryptase beta III
• Tryptase beta2
• Tryptase beta-2
• Tryptase II
• tryptase III
• tryptase-2
• tryptaseB
• tryptaseC
• Tryptase-C

Background

Tryptases are trypsin-like serine proteases, with beta tryptases as the main isoenzymes expressed in mast cells (1). They are stored in secretory granules of mast cells, where they form active tetramers with heparin proteoglycan. Because of the unique arrangement of the active sites in the tetramer, which are facing a narrow central pore, beta tryptases are resistant to macromolecule protease inhibitors (2). When mast cells are activated, beta tryptases are released along with other proteins in secretory granules, participating in provoking inflammatory conditions (3). beta tryptases have been implicated as mediators in the pathogenesis of asthma and other allergic disorders. According to the protein sequence, this product was previously labeled as human Tryptase beta-2 protein upon release.  This product has since been renamed as Recombinant Human Tryptase alpha/beta-1 to reflect current sequence alignment per sequence revisions and classification adjustments that have occurred in the NCBI databases. 

1. Caughey, G. H. 2004, in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. pp. 1535.
2. Sommerhoff, C.P. et al. (1999) Proc. Natl. Acad. Sci. USA. 96:10984.
3. Hallgren, J. and G. Pejler (2006) FEBS J. 273:1871.  

Bioinformatics

• Gene Symbol: TPSB2
• Uniprot:
  • Q15661.1()