Recombinant Human Trypsin 3/PRSS3 Protein, CF Summary
Product Specifications
Val16-Ser247, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
3710-SE
| Formulation | Supplied as a 0.2 μm filtered solution in MES and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0
- Recombinant Human Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3710-SE)
- Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # 1585-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- Substrate: Mca-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(Dnp)-NH2 (Catalog # ES002)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rhEnterokinase with Thermolysin.
- Dilute rhEnterokinase to 100 µg/mL with Activation Buffer.
- Dilute Thermolysin to 3.16 μg/mL with Activation Buffer.
- Mix equal volumes of diluted rhEnterokinase with Thermolysin
- Incubate at 37 °C for 30 minutes.
- Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline.
- Activate rhTrypsin 3, with activated rhEnterokinase.
- Dilute the activated rhEnterokinase to 2 µg/mL in Assay Buffer.
- Dilute rhTrypsin 3 to 200 µg/mL in Assay Buffer.
- Mix equal volumes of the diluted rhEnterokinase and diluted rhTrypsin 3.
- Incubate at 37 °C for 90 minutes.
- After incubation, dilute activated rhTrypsin 3 to 0.1 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load in a black well plate 50 µL of rhTrypsin 3 at 0.1 µg/mL, 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 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- 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).
- rhTrypsin 3: 0.005 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: Trypsin 3/PRSS3
Human Trypsin 3, encoded by the PRSS3 gene, is also known as mesotrypsin (1). Constituting less than 10% of the total trypsin content in normal pancreatic juice, it is one of the three trypsin isoforms produced by the pancreas (2). Compared to Trypsin 1 and 2, one intriguing feature of Trypsin 3 is its resistance to polypeptide trypsin inhibitors, such as the Kunitz-type soybean trypsin inhibitor or the Kazal-type pancreatic secretory trypsin inhibitor. As revealed by the crystal structure, this resistance is likely due to the presence of an arginine residue in place of the highly conserved Gly198 (3). Trypsin 3 is synthesized in the pancreas and secreted into the duodenum lumen, where it is activated by enterokinase. One physiologic function of Trypsin 3 has been proposed to be degradation of trypsin inhibitors, which facilitates the digestion of those foods rich in these proteins (4).
- Nyaruhucha, C.N.M. et al. (1997) J. Biol. Chem. 272:10573.
- Rinderknecht, H. et al. (1984) Gastroenterology 86:681.
- Katona, G. et al. (2002) J. Mol. Biol. 315:1209.
- Szmola, R. et al. (2003) J. Biol. Chem. 278:48580.
Citations for Recombinant Human Trypsin 3/PRSS3 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Mesotrypsin and caspase-14 participate in prosaposin processing: potential relevance to epidermal permeability barrier formation.
Authors: Yamamoto-Tanaka M, Motoyama A, Miyai M, Matsunaga Y, Matsuda J, Tsuboi R, Hibino T
J Biol Chem, 2014-05-28;289(29):20026-38.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion.
Authors: Wu ZL, Ethen C, Hickey GE, Jiang W
Biochem. Biophys. Res. Commun., 2009-01-06;379(3):749-53.
Species: Virus
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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