Recombinant Human Tryptase gamma-1/TPSG1 Protein, CF
Recombinant Human Tryptase gamma-1/TPSG1 Protein, CF Summary
Arg20-Arg281, with a C-terminal 10-His tag
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, CaCl2, NaCl, Brij and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5 (TCNB)
- Assay Buffer: 0.1M Tris, pH 8.0
- Recombinant Human Tryptase gamma ‑1/TPSG1 (rhTPSG1) (Catalog # 1667-SE)
- Trypsin, (Sigma, Catalog # T-1426)
- Recombinant Human Serpin A1/ alpha 1‑Antitrypsin (rhSerpin-A1) (Catalog # 1268-PI)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhTPSG1 to 100 µg/mL in Activation Buffer.
- Dilute Trypsin to 0.4 µg/mL in Activation Buffer.
- Combine equal volumes of 100 µg/mL rhTPSG1 and 0.4 µg/mL Trypsin. Include a control containing Activation Buffer in place of rhTPSG1 (Trypsin control).
- Incubate at 37 °C for 2 hours.
- Dilute rhSerpin-A1 to 18.2 µg/mL in Assay Buffer.
- Add rhSerpin-A1 to the reaction mixture making a one half dilution for final concentrations of 9.1 µg/mL rhSerpin-A1, 25 µg/mL rhTPSG1, and 0.1 µg/mL Trypsin. Addition of rhSerpin-A1 will stop the activity of Trypsin. Control from step three will verify that rhSerpin-A1 is working correctly.
- Incubate all samples for 15 minutes at room temperature.
- Dilute rhTPSG1 to 4 ng/µL in Assay Buffer. Dilute Trypsin control equally.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the diluted samples into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmoles/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhTPSG1: 0.200 µg
- Substrate: 10 µM
Background: Tryptase gamma-1/TPSG1
As mediators of inflammatory and allergic response, mast cells are found throughout the body concentrated near blood vessels in connective tissue and the mucous membranes of the respiratory and gastrointestinal tract (3). Upon activation, human mast cells release granules that are enriched with neutral serine proteases including Tryptases, chymase and cathepsin G (4). Tryptase gamma -1, also called transmembrane Tryptase, is encoded by TPSG1, one of many serine protease genes clustered in human chromosome 16p13.3 (5). Human Tryptase gamma -1 is synthesized as a 321 amino acid preproenzyme with a C-terminal transmembrane anchor (1, 2). The rhTPSG1 was expressed as a soluble protein terminated at residue 281 and corresponded to the proenzyme. The proenzyme can be cleaved by trypsin to form the active enzyme. The serine protease activity of trypsin-activated rhTPSG1 can be inhibited by ecotin (R&D Systems, Catalog # 1328-PI). Greater than 95% protease activity is inhibited by ecotin at approximately 10:1 molar ratio.
- Wong, G.W. et al. (1999) J. Biol. Chem. 274:30784.
- Caughey, G.H. et al. (2000) J. Immunol. 164:6566.
- Harris, J.L. et al. (2001) J. Biol. Chem. 276:34941.
- Miller, H.R.P. and A.D. Pemberton (2002) Immunology 105:375.
- Wong, G.W. et al. (2002) J. Biol. Chem. 277:41906.
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