Enterokinase, Recombinant, Bovine (Enteropeptidase)

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 cytoplamic 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).\n\nSource: A DNA sequence encoding the dipeptide Met-Ala fused to the bovine Enteropeptidase/Enterokinase (Cys 788-His 1035: Accession# P98072) (Kitamoto, Y., et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:7588) was expressed in E. coli.\n\nMolecular Mass: Based on N-terminal amino acid sequencing, the recombinant bovine Enterokinase has two chains, with N-termini starting at Ala-Cys 788 and Ile 801, corresponding to the C-terminal fragment of the heavy chain (Ala-Cys 788-Lys 800) and light chain (Ile 801-His 1035), and are linked by a disulfide bond. The heterodimer predicts a molecular mass of 27.7 kD and migrates as an approximately 30 kD band in SDS-PAGE under non-reducing conditions, The light chain predicts a molecular mass of approximately 26 kD and migrates as an approximately 34 kD band in SDS-PAGE under reducing conditions.\n\nEndotoxin Level: < 1.0 EU per 1 microg of the protein as determined by the LAL method.\n\nActivity: Measured by its ability to cleave the chromogenic substrate, thiobenzyl benzyloxycarbonyl-L- lysinate (Z-Lys-SBzl) in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Cleavage of the substrate was measured by monitoring the rate of 3-carboxy-4-nitrothiophenoxide production from the absorbance changes at 405 nm, using the extinction coefficient 13260 M-1 cm-1. The specific activity, measured with 100 microM substrate and 2 ng enzyme in 50 mM Tris/HCl, pH 7.4 at room temperature, is > 35 nmoles/min/microg. 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 Cat. # T0637 ) to remove the rbEnterokinase from the reaction mixture. SBTI inhibits rbEnterokinase with a K i of 1.6 nM (Lu, D., et al., 1997, J. Biol. Chem. 272:31293).\n\nHandling: Centrifuge the vial before opening to recover entire contents of the vial. Due to possible sublimation during storage, the buffer volume may decrease over time, however, the product is sold by mass and the amount of protein will remain constant. To ensure quantitative recovery, we suggest the stock solution be made in the original vial.\n\nStorage and Stability: Samples are stable for up to six months at -20 degrees C in a manual defrost freezer from the date of receipt. The protein can be aliquoted and stored at -20 degrees C in a manual defrost freezer for three months without a significant loss of activity. Avoid repeated freeze-thaw cycles.

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