Recombinant Bovine Enteropeptidase/Enterokinase Protein, CF

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Product Details

Summary
Reactivity BvSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

Order Details

Recombinant Bovine Enteropeptidase/Enterokinase Protein, CF Summary

Details of Functionality
Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Lys-ThioBenzyl ester (Z-Lys-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Lu, D. et al. (1997) J. Biol. Chem. 272:31293. The specific activity is >35 nmol/min/µg, as measured under the described conditions.
Source
E. coli-derived bovine Enteropeptidase/Enterokinase protein
Cys788-Lys800 (heavy chain C-terminal fragment) with an N-terminal Ala, & Ile801-His1035 (light chain)
Accession #
N-terminal Sequence
Ala & Ile801
Structure / Form
Disulfide-linked heterodimer
Protein/Peptide Type
Recombinant Enzymes
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
1.5 kDa (heavy chain C-terminal fragment), 26 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
34 kDa, reducing conditions
30 kDa, non-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 Glycerol, NaCl and HEPES.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
  • Assay Buffer: 50 mM Tris, pH 7.5
  • Recombinant Bovine Enteropeptidase/Enterokinase (rbEnterokinase) (Catalog # 4139-SE)
  • Substrate: Z-Lys-SBZL (Bachem, Catalog # M-1300), 10 mM stock in DMSO
  • 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
  • 96 well Clear Plate (Costar, Catalog #  92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rbEnterokinase to 0.04 µg/mL in Assay Buffer.
  2. Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
  3. Load into a 96 well clear plate 50 µL of the diluted rbEnterokinase. For a Substrate Blank, load 50 µL of the Assay Buffer.
  4. Start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells.
  5. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  6. Calculate specific activity:

     Specific Activity (nmol/min/µg) =

Adjusted Vmax* (OD/min) x well volume (L) x 109 nmol/M
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:
  • rbEnterokinase: 0.002 µg
  • DTNB: 100 µM
  • Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Bovine 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

EK initiates activation of pancreatic proteases by converting trypsinogen to trypsin, which in turn activates chymotrypsin, carboxypeptidases and elastases. Located in intestinal brush border, it is a disulfide bond linked dimer of the heavy and light chains, which are derived from the same single-chain precursor. The multidomain‑containing heavy chain consists of a short cytoplasmic tail, a transmembrane, a SEA, a SRCR, a MAM, two CUB and two LDL-receptor class A domains. The light chain contains the catalytic domain of trypsin-like serine proteases. The purified recombinant bovine EK (residues 788-1035) corresponds to a disulfide bond‑linked dimer that consists of the C-terminal fragment of the heavy chain (residues 788-800) and the light chain (residues 801-1035). rbEnterokinase can cleave fusion proteins having an accessible Enterokinase cleavage site (DDDDK). At an average ratio for fusion protein:rbEnterokinase of 1000:1 (w/w), cleavage up to 90% completion is achieved within one hour at room temperature. Non-specific cleavage at basic residues has also been observed for some proteins. It is recommended that cleavage reaction be optimized for each fusion protein. The reaction may be terminated by passing the sample through a soybean trypsin inhibitor (SBTI)-agarose affinity column (e.g. Sigma Catalog # T0637 ) to remove the rbEnterokinase from the reaction mixture. SBTI inhibits rbEnterokinase with a Ki of 1.6 nM.

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