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物种
Human分子别名
Enteropeptidase, ENTK, hEK, hEK-His表达宿主
E.coli分子量
28 kDa (Reducing)
纯度
＞95% by SDS-PAGE活性
5 U/μL缓冲体系
20 mM Tris-HCl, 200 mM NaCl, 2 mM CaCl2 , 50% Glycerol(pH 7.4 @ 25°C)
储存条件
Store at -25 ~ -15℃ for 2 years
文献引用
1.Melicherová, Kristína,Krahulec, Ján, afránek, Martin, et al. Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris[J]. Appl Microbiol Biotechnol, 2017, 101(5):1927-1934.
2. Gasparian M E, Ostapchenko V G, Schulga A A, et al. Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli[J]. Protein Expression & Purification, 2003, 31(1):133-139.
Enterokinase is a specific protease that cleaves after lysine at its cleavage site Asp-Asp-Asp-Asp-Lys. It will sometimes cleave at other basic residues, depending on the conformation of the protein substrate. This product is a high-purity, high-specific activity enterokinase prepared using a recombinant E.coli. It boasts a broad range of applicability (operating temperature: 4-45°C; pH range: 4.5-9.5) and retains partial activity in the presence of various detergents and denaturants.
Storage Solution: 5 U/μL hEK-His in 20 mM Tris-HCl, 200 mM NaCl, 2 mM CaCl2 , 50% Glycerol(pH 7.4 @ 25°C)
Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows:
Combine 500 ug of sample with reaction buffer
Recommended Reaction Buffer: 20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 (pH 8.0)
Add 1 U of Enterokinase light chain
Incubate at 25°C for 16 hours
1. Enterokinase is inhibited by high salt concentrations. For optimal activity NaCl concentration should be 50 mM or less. The pH of the buffer should be between 6 and 9. The enzyme requires 2 mM Calcium for activity.
- One unit is defined as the amount of enzyme required to cleave 500 µg of substrate to 95% completion in 16 hours at 25°C.
生物活性
The results of 100μg substrate digestion separated under diferent quantity of hEK-his, the reaction was incubated for 16 h at 25°C.
M marker
Lane 1 Substrate/ hEK-his = 500/1
Lane 2 Substrate/ hEK-his = 1000/1
Lane 3 Substrate/ hEK-his = 2000/1
Lane 4 Substrate/ hEK-his = 3000/1
Lane 5 Substrate/ hEK-his = 4000/1
Lane 6 Substrate/ hEK-his = 5000/1
Lane 7 Substrate/ hEK-his = 6000/1
(1 U hEK-his)
Lane 8 Substrate/ hEK-his = 8000/1
Lane 9 Substrate/ hEK-his = 10000/1
Lane 10 Only substrate
电泳
1μg (R: reducing condition, N: non-reducing condition).
体积排阻色谱(SEC-HPLC)
93%
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