Recombinant Human Enteropeptidase/Enterokinase Protein, CF

Recombinant Human Enteropeptidase/Enterokinase (Catalog # 10438-SE) is measured by its ability to cleave a colorimetric peptide substrate, Z-Lys-SBzl.

2 μg/lane of Recombinant Human Enteropeptidase/Enterokinase (Activated) (Catalog # 10438-SE) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining, showing bands at approximately 110, 100, & 47 kDa under reducing conditions.

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

Recombinant Human Enteropeptidase/Enterokinase Protein, CF Summary

• Additional Information: Activated
• Details of Functionality: Measured by its ability to cleave a colorimetric peptide substrate, Z-Lys-SBzl. The specific activity is >30,000 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Enteropeptidase/Enterokinase protein
  Leu41-His1019
  with a C-terminal 9-His tag
• Accession #: P98073
• N-terminal Sequence: Leu178, Ala294, Ile306 (heavy chain) & Ile785 (light chain)
• Structure / Form: Activated, heavy and light chains
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 67, 55, 53 kDa (heavy chain) & 27 kDa (light chain).
  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: 98-117 kDa (heavy chain) & 46-49 kDa (light chain), reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl₂.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
  • Recombinant Human Enteropeptidase/Enterokinase, active (rhEnterokinase) (Catalog # 10438-SE)
  • Substrate: thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) (Bachem, Catalog # M-1300), 10 mM stock in DMSO
  • 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D8130), 10 mM stock in DMSO
  • 96-well clear plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhEnterokinase to 0.04 µg/mL in Assay Buffer.
  2. Dilute Substrate to 400 µM in Assay Buffer containing 400 µM of DTNB.
  3. In a plate, load 50 µL of the diluted rhEnterokinase, and start the reaction by adding 50 µL of Substrate/DTNB mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate/DTNB mixture.
  4. Read at an absorbance of 405 nm in kinetic mode of 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 13260 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD

  Per Well:
  • rhEnterokinase: 0.002 µg
  • DTNB: 200 µM
  • Substrate: 200 µM

Notes

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

Alternate Names for Recombinant Human Enteropeptidase/Enterokinase Protein, CF

• EC 3.4.21
• EC 3.4.21.9
• Enterokinase
• Enteropeptidase
• ENTK
• ENTKenterokinase
• MGC133046
• protease, serine, 7 (enterokinase)
• PRSS7
• PRSS7enteropeptidase
• Serine protease 7
• TMPRSS15
• Transmembrane protease serine 15
• transmembrane protease, serine 15

Background

Enteropeptidase, also known as enterokinase, is a type II transmembrane serine protease that initiates activation of pancreatic proteases by converting trypsinogen to trypsin, which subsequently leads to activation of chymotrypsin, carboxypeptidases and elastases in the intestine (1). Located in the intestinal brush border, enteropeptidase is a disulfide bond-linked dimer of an N-terminal heavy chain (HC) and C-terminal light chain (LC) derived from the same single-chain precursor. The non-catalytic multidomain HC includes a short cytoplasmic tail, a transmembrane, a MSCR, a MAM, two CUB, and two LDL-receptor class A domains while the LC contains the catalytic domain of trypsin-like serine proteases (1,2). Enteropeptidase is known to have high sequence specificity making it useful as a biotechnological tool for recombinant fusion domains. Human enteropeptidase LC has greater efficiency and specificity than bovine enteropeptidase LC (3,4). Removal of HC domains results in significant loss of activity towards its physiological substrate trypsinogen (5-7) although cleavage of small peptidyl substrates remains equivalent (6,8). Enteropeptidase inhibition may be a target in diabetes and obesity (9,10). The purified activated recombinant human Enteropeptidase corresponds to the heterodimer of LC and HC without the transmembrane domain.

1. Zheng, X.L. et al. (2009) Front. Biosci. 1:242.
2. Lu, D. et al. (1999) J. Mol. Biol. 292:361.
3. Gasparian, M.E. et al. (2006) Biochemistry 71:113.
4. Mikhailova, A.G. et al. (2007) Protein Pept. Lett. 14:227.
5. LaVallie, E.R. et al. (1993) J. Biol. Chem. 268:23311.
6. Lu, D. et al. (1997) J. Biol. Chem. 272:31293.
7. Mikhailova, A.G. et al. (1999) FEBS Lett. 442:226.
8. Light, A and P Fonseca. (1984) J. Biol. Chem. 259:13195.
9. Braud, S. et al. (2012) PLoS One 7:e49612.
10. Yashiro, H. et al. (2019) Diabetes Obes. Metab. 21:2228.

Publications for Enteropeptidase/Enterokinase (10438-SE)(1)

Publication using 10438-SE

• Hoffka, G;Mhana, S;Vas, M;Toldi, V;Mótyán, JA;T?zsér, J;Szabó, A; Modeling protease-sensitive human pancreatic lipase mutations in the mouse ortholog The Journal of biological chemistry 2024-09-10 [PMID: 39265662] (Bioassay, E. coli)

Bioinformatics

• Uniprot:
  • P98073()