A DNA sequence encoding the light chain (Ile 801-His 1035) of bovine Enterokinase(NP_776864)was expressed.
Yeast (Pichia pastoris)
> 95%, as determined by SDS-PAGE and SEC-HPLC Analysis
< 1.0 EU per 1 μg of the cytokine as determined by the LAL method
Samples are stable for up to twelve months from date of receipt -70℃
Predicted N terminal:
One unit is defined as the amount of enzyme needed to cleave 50μg fusion protein in 16 hours at 20℃ in a buffer containing 20 mM Tris-HCl, 50 mM NaCl, and 2 mM CaCl2>, pH 7.4.
The recombinant bovine Enterokinase consists of 234 amino acids and has a calculated molecular mass of 26.1kDa. As a result of glycosylation, the recombinant protein migrates as an approximately 45kDa protein in SDS-PAGE under reducing conditions.
Shipped at room temperature. The liquid or lyophilized enzyme is stable for at least 21d when stored at 37 ℃ (or room temperature). Store it under sterile conditions at -70℃ upon receiving. Recommend to aliquot the protein into smaller quantities for optimal storage.Avoid repeated freeze-thaw cycles.
Resuspend the enzyme powder with sterile water. Keep reconstituted enzyme at -20℃ in aliquots.
An example to estimate the appropriate usage of the enzyme:
Each target protein presents the cleavage site somewhat differently, it is recommended to test several enterokinase concentrations, temperatures and incubation times to optimize specificity and efficiency of cleavage. The following protocol is an example to estimate appropriate conditions.
Examples for cleavage of fusion proteins:
Enterokinase is a member of the trypsin family of serine proteases. The precursor protein is cleaved into two chains which then forms a heterodimer linked by a disulfide bond. The heavy chain anchors enterokinase in the intestinal brush border membrane and the light chain is the catalytic subunit, which initiates conversion activation of a subset of pancreatic proteolytic proenzymes. Enterokinase is the physiological activator of trypsinogen and has a specificity for the sequence (Asp)4-Lys-Ile. The mature trypsin in turn activates other proenzymes including chymotrypsinogen, procarboxypeptidases, and proelastases. In addition, Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins.
Light, A. et al., 1989, Trends Biochem. Sci. 14: 110-112.
Kitamoto, Y. et al., 1994, Proc. Natl. Acad. Sci. 91: 7588-7592.
Lavallie, E.R. et al., 1993, J. Biol. Chem. 268: 23311-23317.
Collins-Racie, L.A. et al., 1995, Biotechnology. 13: 982-957.
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