Enterokinase Protein, Bovine (P.pastoris, His)

Enterokinase Protein, Bovine (P.pastoris, His) is a glycosylated peptide chain with a His-tag produced in P. pastoris. Enterokinase Protein is a serine protease that plays a crucial role in the digestive system. By specifically cleaving trypsinogen to convert it into active trypsin, Enterokinase Protein initiates the cascade activation of pancreatic zymogens. Enterokinase Protein serves as a key initiator of the intestinal digestive enzyme cascade^[1][2][3].

Enterokinase is a glycoprotein mainly present in the duodenal and jejunal mucosa. The core role of enterokinase is to activate digestive zymogens, converting trypsinogen into trypsin. The activated trypsin can not only directly decompose proteins but also further activate other zymogens such as chymotrypsinogen, procarboxypeptidase, and proelastase, forming a digestive enzyme cascade reaction to promote protein digestion^[1][2][3].

100 IU/μg Unit Definition: One unit is defined as the amount of enzyme needed to cleave 50 μg of fusion protein in 16 hours to 95% completion at 22°C in a buffer containing 25mM Tris-HCl, pH 8.0.

Materials
Test protein: Enterokinase Protein, Bovine (P.pastoris, His) (EK, HY-P7341)
100 μg Cleavage Control Protein
Storage buffer: 20 mM Tris HCl, pH 7.4, 200 mM NaCl, 2 mM CaCl₂, 50% glycerol
Capture buffer: 200 mM Tris HCl, pH 7.4, 500 mM NaCl, 20 mM CaCl₂

Digestion preparation
1. For the small scale cleavage, EK can be diluted by EK Storage Buffer directly.
2. In order to get a fully dissolved EK for the scaled up reaction, reconstitute the lyophilized EK powder in sterile deionized water at 1mg/ml first. Then dilute it in EK Dilution/Storage Buffer according to the requirement.
3. The dilutions can be stored in dilution buffer at 20°C for several weeks. To avoid loss of activity, extended storage of dilutions is not recommended.

EK Digestion
Each target protein has a different position of cleavage site. Therefore, it is recommended to conduct small scale reactions first to optimize the concentration of EK, incubation time and temperature.
Small scale reaction proposal
1. Make 4 appropriate serial dilutions of EK vs. target protein (w/w), for example 1:50; 1:100; 1:200; 1:400. For 10 μg fusion protein cleavage, the correspondent EK quantity is 0.2 μg; 0.1 μg; 0.05 μg; 0.025 μg.
2. Assemble the following components in labeled tubes.
3. Incubate the reactions at room temperature (20°C-25°C).
4. Take 10 μL aliquots into SDS PAGE sample buffer after 2, 4, 8 and 16 h.
5. Determine the extent of cleavage of the samples by SDS PAGE analysis.
Scale up
Scale up the reaction proportionally according to the best cleavage result.
Caution:If the buffer of fu sion protein contains the following reagents, it is necessary to dialyze your fusion protein before the digestion.
1. > 2 M urea, > 250 mM NaCl, > 20 mM β-mercaptoethanol, >0.1% SDS, >1% Triton X-100, or > 50 mM imidazole.
2. pH values below 6 and above 9.
3. Avoid the presence of serine protease inhibitors.

Bovine

P. pastoris

C-His

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Approximately 40 kDa, based on SDS-PAGE under reducing conditions.

≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder

Lyophilized from a 0.22 μm filtered solution of 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2 mM CaCl₂.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<0.2 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 1 mg/mL in sterile EK Storage Buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2 mM CaCl₂, 50% glycerol).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.

• [1]. Light A, et al. The amino-terminal sequence of the catalytic subunit of bovine enterokinase. J Protein Chem. 1991 Oct;10(5):475-80.  [Content Brief]

  [2]. Mann NS, et al. Enterokinase. Proc Soc Exp Biol Med. 1994 Jun;206(2):114-8.  [Content Brief]

  [3]. Kitamoto Y, et al. Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7588-92.  [Content Brief]