• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH    ₂ (Catalog # ). The specific activity is >17,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Phe16-Asn246, with a C-terminal 10-His tag Accession # NP_035775

• Accession #

• N-terminal Sequence    
  Analysis

  Phe16

• Structure / Form

  Pro form

• Predicted Molecular Mass

  26 kDa

• SDS-PAGE

  29 kDa and 36 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)

• Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 8.0

• Recombinant Mouse Trypsin 3/PRSS3 (rmTrypsin 3) (Catalog # 3565-SE)

• Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # )

• Bacterial Thermolysin (Thermolysin) (Catalog # )

• 1,10 Phenanthroline (Sigma, Catalog # 320056) 0.6 M in DMSO

• Substrate MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ), 2 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rhEnterokinase with Thermolysin.

1. Dilute rhEnterokinase to 100 µg/mL in Activation Buffer.

2. Dilute Thermolysin to 3.16 µg/mL in Activation Buffer.

3. Mix equal volumes of diluted rhEnterokinase and Thermolysin.

4. Incubate at 37 °C for 30 minutes.

5. Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline to the reaction tube.

2. Activate rmTrypsin 3 with activated rhEnterokinase.

1. Dilute activated rhEnterokinase to 2 µg/mL in Assay Buffer.

2. Dilute rmTrypsin 3 to 200 µg/mL in Assay Buffer.

3. Mix equal volumes of activated rhEnterokinase and rmTrypsin 3.

4. Incubate at room temperature for 1 hour.

3. Dilute activated rmTrypsin3 to 0.04 µg/mL in Assay Buffer.

4. Dilute Substrate to 20 µM in Assay Buffer.

5. Load 50 µL of 0.04 µg/mL of rhTrypsin 3 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 20 µM Substrate.

6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.

7. Calculate specific activity: 

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rmTrypsin 3: 0.002 µg

• Substrate: 10 µM

Background: Trypsin 3/PRSS3

Mouse Trypsin-3, encoded by the PRSS3 gene, is also known as mesotrypsin. It consists of a signal peptide (residues 1 to 15), a pro region (residues 16 to 23), and a mature chain (residues 24 to 246) (1). The purified recombinant mouse Trypsin 3 corresponds to the pro form, which can be activated by enterokinase.

• References:

1. Halfon, S. et al. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) pp. 1483, Academic Press, San Diego.

• Entrez Gene IDs:

  5646 (Human); 22073 (Mouse); 362347 (Rat)

• Alternate Names:

  Brain trypsinogen; EC 3.4.21; EC 3.4.21.4; Mesotrypsin; mesotrypsinogen; MTG; pancreatic trypsinogen III; protease, serine, 3 (mesotrypsin); protease, serine, 3; protease, serine, 4 (trypsin 4, brain); PRSS3; PRSS4; Serine protease 3; Serine protease 4; T9; TRY3MTG; TRY4mesotrypsin; Trypsin 3; Trypsin 4; Trypsin III; Trypsin IV; trypsin-3; Trypsinogen 15; trypsinogen 4; trypsinogen 5; trypsinogen IV