Recombinant Bovine

Enteropeptidase/Enterokinase Catalog Number: 4139-SE

DESCRIPTION Source E. coli­derived Cys788­Lys800 (heavy chain C­terminal fragment) with an N­terminal Ala, & Ile801­His1035 (light chain) Accession # P98072

N­terminal Sequence Ala & Ile801 Analysis Structure / Form Disulfide­linked heterodimer

Predicted Molecular 1.5 kDa (heavy chain C­terminal fragment), 26 kDa (light chain) Mass

SPECIFICATIONS SDS­PAGE 34 kDa, reducing conditions 30 kDa, non­reducing conditions

Activity Measured by its ability to cleave a colorimetric peptide substrate, N­carbobenzyloxy­Lys­ThioBenzyl ester (Z­Lys­SBzl), in the presence of 5,5’Dithio­bis (2­nitrobenzoic 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.

Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method.

Purity >90%, by SDS­PAGE under reducing conditions and visualized by silver stain. Formulation Supplied as a 0.2 μm filtered solution in Glycerol, NaCl and HEPES. See Certificate of Analysis for details.

Activity Assay Protocol Materials l Assay Buffer: 50 mM Tris, pH 7.5 l Recombinant Bovine Enteropeptidase/Enterokinase (rbEnterokinase) (Catalog # 4139­SE) l Substrate: Z­Lys­SBZL (Bachem, Catalog # M­1300), 10 mM stock in DMSO l 5,5’­dithio­bis (2­nitrobenzoic acid) (DTNB) (Sigma, Catalog # D­8130), 10 mM stock in DMSO l 96 well Clear Plate (Costar, Catalog # 92592) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Assay 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: Adjusted V * (OD/min) x well volume (L) x 109 nmol/M Specific Activity (nmol/min/µg) = max ext. coeff** (M­1cm­1) x path corr.*** (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank **Using the extinction coefficient 13260 M­1cm­1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD Final Assay Per Well: Conditions l rbEnterokinase: 0.002 µg l DTNB: 100 µM l Substrate: 100 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 freeze­thaw cycles. l 6 months from date of receipt, ­20 to ­70 °C as supplied. l 3 months, ­20 to ­70 °C under sterile conditions after opening.

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 single­chain precursor. The multidomain­ containing heavy chain consists of a short cytoplasmic tail, a transmembrane, a SEA, a SRCR, a MAM, two CUB and two LDL­receptor class A domains. The light chain contains the catalytic domain of trypsin­like serine proteases. The purified recombinant bovine EK (residues 788­1035) corresponds to a disulfide bond­linked dimer that consists of the C­terminal fragment of the heavy chain (residues 788­800) and the light chain (residues 801­1035). 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. Non­specific 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.

Rev. 2/6/2018 Page 1 of 1