Recombinant Mouse Testisin/Prss21 Protein, CF

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

Recombinant Mouse Testisin/Prss21 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a flourogenic peptide substrate Pro-Phe-Arg-7-amido-4-methylcoumarin (PFR-AMC). The specific activity is >330 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Testisin/Prss21 protein
  Leu22-Asn298, with a C-terminal 10-His tag
• Accession #: Q9JHJ7
• N-terminal Sequence: Leu22
• Protein/Peptide Type: Recombinant Proteins
• Gene: Prss21
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 33 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: 46-50 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 MES, NaCl, CaCl₂ and Brij-35.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, pH 7.5 (TCN)
  • Recombinant Mouse Testisin/Prss21 (rmTestisin) (Catalog # 6820-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1, 10-phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • Substrate: Pro-Phe-Arg-AMC (Bachem, Catalog # I-1295), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmTestisin to 200 ng/μL in Assay Buffer.
  2. Dilute Thermolysin to 0.2 μg/mL in Assay Buffer.
  3. Combine equal volumes of the diluted rmTestisin and diluted Thermolysin and incubate at 37 °C for 30 minutes to activate rmTestisin.
  4. After incubation, stop reaction by adding an equal volume of 20 mM 1, 10-phenanthroline for a final concentration of 10 mM.
  5. Dilute activated rmTestisin to 1 ng/μL in Assay Buffer.
  6. Dilute Substrate to 200 μM in Assay Buffer.
  7. Load into a plate 50 μL of 1 ng/μL activated rmTestisin, and start the reaction by adding 50 μL of 200 μM Substrate. For a Substrate Blank, load 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.
  8. Read plate at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes.
  9. 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 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9891)

  Per Well:
  • rmTestisin: 0.050 μg
  • 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 Testisin/Prss21 Protein, CF

• EC 3.4.21
• Eosinophil serine protease 1
• ESP1EC 3.4.21.-
• ESP-1serine protease from eosinophils
• protease, serine, 21 (testisin)
• Prss21
• Serine protease 21
• TEST1
• TEST1testisin
• Testisin

Background

Testisin is a serine protease encoded by the PRSS21 gene. Testisin is similar in structure to tryptases, and is also known as Tryptase‑4 (1). It is predicted to have substrate specificity similar to that of trypsin (2). Testisin is most highly expressed in testicular germ cells, but is also present in eosinophils (3). Testisin is known to be required for spermatogenesis, and deficiencies of the enzyme can result in infertility (4, 5). Testisin is anchored to the cell surface through a C-terminal glycosylphosphatidylinositol anchor (6). Recombinant mouse Testisin is expressed with a truncated C-terminus, resulting in its secretion. The recombinant protein was purified as the zymogen and can undergo autoactivation under the appropriate conditions.

1. Wong, G.W. et al. (2001) J. Biol. Chem. 276:20648.
2. Hooper, J.D. et al. (2000) Biochim. Biophys. Acta 1492:63.
3. Inoue, M. et al. (1998) Biochem. Biophys.  Res. Comm. 252:307.
4. Netzel-Arnett, S. et al. (2009) Biol. Reprod. 81:921.
5. Kawano, N. et al. (2010) Biol. Reprod. 83:359.
6. Honda, A. et al. (2002) J. Biol. Chem. 277:16976.

Bioinformatics

• Gene Symbol: Prss21
• Uniprot:
  • Q9JHJ7()