Recombinant Bovine Enteropeptidase/Enterokinase Protein, CF
Recombinant Bovine Enteropeptidase/Enterokinase Protein, CF Summary
Product Specifications
Cys788-Lys800 (heavy chain C-terminal fragment) with an N-terminal Ala, & Ile801-His1035 (light chain)
Analysis
30 kDa, non-reducing conditions
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.
4139-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Glycerol, NaCl and HEPES. |
| 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
- 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
- Dilute rbEnterokinase to 0.04 µg/mL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
- Load into a 96 well clear plate 50 µL of the diluted rbEnterokinase. For a Substrate Blank, load 50 µL of the Assay Buffer.
- Start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells.
- Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
- 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
Reconstitution Calculator
Background: Enteropeptidase/Enterokinase
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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