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Recombinant Human Enteropeptidase/Enterokinase (Activated) Catalog Number: 10438-SE

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Recombinant Human Enteropeptidase/Enterokinase (Activated) Catalog Number: 10438-SE Recombinant Human Enteropeptidase/Enterokinase (Activated) Catalog Number: 10438-SE DESCRIPTION Source Mouse myeloma cell line, NS0-derived human Enteropeptidase/Enterokinase protein Leu41-His1019 Accession # P98073 with a C-terminal 9-His tag N-terminal Sequence Leu178, Ala294, Ile306 (heavy chain) & Ile785 (light chain) Analysis Structure / Form Activated, heavy and light chains Predicted Molecular 67, 55, 53 kDa (heavy chain) & 27 kDa (light chain) Mass SPECIFICATIONS SDS-PAGE 98-117 kDa (heavy chain) & 46-49 kDa (light chain), reducing conditions Activity 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. Endotoxin Level <0.10 EU per 1 μg of the protein by the LAL method. Purity >95%, by SDS-PAGE under reducing conditions and visualized by silver stain. Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2. See Certificate of Analysis for details. Activity Assay Protocol Materials 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 Assay 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: 12 Adjusted Vmax * (OD/min) x well volume (L) x 10 pmol/mol Specific Activity (pmol/min/µg) = 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 Final Assay Per Well: Conditions rhEnterokinase: 0.002 µg DTNB: 200 µM Substrate: 200 µM PREPARATION AND STORAGE 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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening. DATA Rev. 3/4/2020 Page 1 of 2 Recombinant Human Enteropeptidase/Enterokinase (Activated) Catalog Number: 10438-SE Enzyme Activity SDS-PAGE Recombinant Human 2 μg/lane of Recombinant Human Enteropeptidase/Enterokinase Enteropeptidase/Enterokinase (Activated) (Catalog # 10438-SE) was resolved with SDS-PAGE (Catalog # 10438-SE) is under reducing (R) and non-reducing (NR) conditions and measured by its ability to cleave visualized by silver staining, showing bands at approximately 110, a colorimetric peptide substrate, 100, & 47 kDa under reducing conditions. Z-Lys-SBzl. 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. References: 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. Rev. 3/4/2020 Page 2 of 2 .
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