Recombinant Human Enteropeptidase/Enterokinase Protein, CF
Recombinant Human Enteropeptidase/Enterokinase Protein, CF Summary
Product Specifications
Leu41-His1019
with a C-terminal 9-His tag
Analysis
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.
10438-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2. |
| 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, 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
- Dilute rhEnterokinase to 0.04 µg/mL in Assay Buffer.
- Dilute Substrate to 400 µM in Assay Buffer containing 400 µM of DTNB.
- 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.
- Read at an absorbance of 405 nm in kinetic mode of 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Vmax* (OD/min) x well volume (L) x 1012 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
- rhEnterokinase: 0.002 µg
- DTNB: 200 µM
- Substrate: 200 µM
Scientific Data
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Recombinant Human Enteropeptidase/Enterokinase (Catalog # 10438-SE) is measured by its ability to cleave a colorimetric peptide substrate, Z-Lys-SBzl.
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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.
Reconstitution Calculator
Background: Enteropeptidase/Enterokinase
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.
- Zheng, X.L. et al. (2009) Front. Biosci. 1:242.
- Lu, D. et al. (1999) J. Mol. Biol. 292:361.
- Gasparian, M.E. et al. (2006) Biochemistry 71:113.
- Mikhailova, A.G. et al. (2007) Protein Pept. Lett. 14:227.
- LaVallie, E.R. et al. (1993) J. Biol. Chem. 268:23311.
- Lu, D. et al. (1997) J. Biol. Chem. 272:31293.
- Mikhailova, A.G. et al. (1999) FEBS Lett. 442:226.
- Light, A and P Fonseca. (1984) J. Biol. Chem. 259:13195.
- Braud, S. et al. (2012) PLoS One 7:e49612.
- Yashiro, H. et al. (2019) Diabetes Obes. Metab. 21:2228.
Citation for Recombinant Human Enteropeptidase/Enterokinase Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Modeling protease-sensitive human pancreatic lipase mutations in the mouse ortholog
Authors: Hoffka, G;Mhana, S;Vas, M;Toldi, V;Mótyán, JA;T?zsér, J;Szabó, A;
The Journal of biological chemistry
Species: E. coli
Sample Types: Whole Cells
Applications: Bioassay
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