Abstract
Enterokinase (EC 3.4.21.9), also known as enteropeptidase, is a highly specific serine protease that plays a critical role in both digestive biology and modern biotechnology workflows. Originally discovered over a century ago, its unique specificity for a defined recognition sequence has made it a valuable tool in protein purification and engineering.
Enterokinase was first described in 1902 by Eugen von Knauer, who observed that an intestinal extract could activate the zymogen trypsinogen into active trypsin. The responsible agent, later named enterokinase, was found to originate from the duodenal brush border and function as the initiator of the entire digestive enzyme cascade.
Structure and Specificity: Enterokinase is a type II transmembrane glycoprotein composed of two chains.
- Heavy Chain (~100–140 kDa): Anchors the enzyme to the membrane and plays a role in substrate recognition.
- Light Chain (~35 kDa): Contains the catalytic serine protease domain, homologous to trypsin.
Recombinant Enterokinase: Precision Cleavage for N-Terminal Fusion Tags
Recombinant enterokinase (rEK) is the light chain subunit of bovine intestinal enterokinase, engineered for high purity and performance. It retains the same cleavage specificity as its native counterpart, precisely recognizing and cleaving at the sequence Asp-Asp-Asp-Asp-Lys↓ (DDDDK). This enables efficient removal of N-terminal fusion tags from recombinant proteins—making rEK a preferred protease in protein purification workflows.
One of rEK’s key advantages is its minimal off-target activity, reducing the risk of non-specific cleavage on similar acidic sequences.
Featured Product: Yeasen Recombinant Bovine Enterokinase, His-tagged, Yeast-expressed
Yeasen’s Recombinant Bovine Enterokinase is a high-purity, high-specificity protease expressed via Pichia pastoris secretion. Engineered for robust performance and scalable production, this His-tagged version is ideal for precise removal of N-terminal fusion tags in recombinant protein workflows.
- Exceptional Specificity: Precisely cleaves after the recognition sequence Asp-Asp-Asp-Asp-Lys↓ (DDDDK), with no detectable off-target activity.
- Ultra-Pure Preparation: Free from contaminating proteases or non-specific cleavage—ensuring clean and reliable results.
- Animal-Origin Free: Fully recombinant production, with no animal-derived raw materials or risk of exogenous viral contamination—ideal for sensitive or regulated applications.
- Consistent Quality: Manufactured in controlled batches to ensure lot-to-lot consistency and stable enzymatic activity across production cycles.
- Scalable Supply: With access to 5L to 1500L fermentation systems, Yeasen can meet product demands at all scales—from R&D to large-scale commercial production. A 500L fermenter yields approximately 50 million units of active enterokinase.
- Flexible Grade Options: Available in both, R&D Grade (Cat# 20395ES) and GMP Grade (Cat# 20396ES), manufactured under ISO 13485 quality management system for clinical and regulatory use.
Performance Demonstration
Enterokinase activity test:
Figure 1. The enzyme cutting effect of Yeasen enterokinase (Cat#20395ES) is consistent with Competitor N*
【Note】: The positive substrate (Cat#20391ES) is a fusion protein composed of two specific protein sequences connected by DDDDK (enterokinase recognition and cutting site), with an overall molecular weight of about 64.6 kDa, which can be specifically cut into two independent protein fragments by enterokinase, with molecular weights of about 27.9 kDa and 36.6 kDa, respectively. This product, as an enterokinase substrate, can be used for semi-quantitative or qualitative enzyme activity detection of recombinant or natural enterokinase.
Enterokinase purity test
Figure 2. The purity of enterokinase (Cat#20395ES) is greater than 95%
【Note】: The theoretical value of enterokinase is 22.7 kDa. Due to the glycosylation effect after expression in Pichia pastoris, the SDS-PAGE shows that the molecular weight of the target protein is about 40 kDa. Generally, the glycosylated bands will have a slightly diffuse band running upwards (as shown in the right figure). Our company can also provide deglycosylation enzyme Endo H (Cat#20414) for customers to deglycosylate and run gel to measure purity (as shown in the left figure).
Enterokinase cutting specificity test
Figure 3. Yeasen enterokinase (20395ES) has less non-specific cutting than import Competitor N
【Note】: Under the same conditions, Yisheng enterokinase (20395ES) and import competitor N's enterokinase were used for enzyme cutting tests. The results show that after purification, the non-specific cutting impurities produced by Yisheng enterokinase (20395ES) are significantly lower than those of import competitor N.
Enterokinase repeated freeze-thaw, accelerated stability test:
Figure 4. Yeasen enterokinase (Cat#20395ES) repeated freeze-thaw, accelerated stability test shows that enzyme activity did not change significantly.
Note: Yeasen enterokinase (Cat#20395ES) randomly selected 2 batches, repeated freeze-thaw 10 times, 20 times, enzyme activity did not change significantly from the initial enzyme activity.
Storage at 25°C for 7, 16, 32 days, 37°C for 7, 14 days, enzyme activity did not change significantly from the initial enzyme activity.
UCF.ME: Yeasen GMP Grade Enzyme Production Base
ISO Certified Ultra-Clean Molecular Enzyme Production Base-UCF.ME, equipped with industrial-grade AKTA purification and physicochemical analysis equipment, hundred-liter high-density fermentation units, ten-thousand-grade standard clean workshops, and automated production lines. It possesses fermentation and purification systems of various specifications, including 5L, 10L, 30L, 100L, 500L, and 1,500L, with the capability to scale up production from 5L to 1,500L in the field of molecular enzyme industrialization, fully meeting the core enzyme raw material needs of downstream customers at different stages such as research and development, pilot scale, and large-scale production.
Yeasen has a strong R&D team and advanced enzyme production base, continuously improving the quality and capacity of key enzymes for recombinant enterokinase through continuous technological innovation and optimization, offering gradient pricing for different specifications to meet the various needs of customers at different production stages.
Product information
|
Product Name |
Product Number |
Specification |
|
20395ES60 20395ES76 20395ES90 20395ES92 20395ES94 |
100 U 500 U 5000 U 100 KU 1 MU(1000 KU) |
Related products
|
Product Name |
Product Number |
Specification |
|
20391ES03/11 |
1 mg/ 4 mg |
|
|
20418ES60 |
100 μg |
|
|
20417ES03/10 |
1 mg/10 mg |
|
|
20414ES92/97 |
10000 U/50000 U |