Recombinant Enterokinase is a high-purity recombinant fragment of bovine enterokinase light chain, with an amino acid sequence identical to that of the bovine enterokinase light chain. It has the same specific cleavage site as naturally extracted enterokinase, which is Asp-Asp-Asp-Asp-Lys (DDDDK). It can be used to remove fusion proteins located at the N-terminus of proteins to remove unwanted fusion tags, ensuring the accurate N-terminal sequence of recombinant fusion proteins. At the same time, Recombinant Enterokinase has higher cutting activity than natural enzymes.

Recombinant Enterokinase, His Tag is a high-purity, high-activity, and highly specific bovine enterokinase expressed by Pichia pastoris secretion. It can effectively cleave fusion proteins within a wide pH range (4.5-9.5) and a wide temperature range, and still has partial activity under various detergents and denaturants. This product has a His tag, which can be easily removed through Ni²⁺ affinity column after the cutting reaction, greatly simplifying the subsequent purification process.

Features

• Strong specificity—A specific protease that cuts the carboxyl terminus of lysine containing four aspartic acids in front: Asp-Asp-Asp-Asp-Lys.
• High purity—No other protease,No non-specific cutting.
• Animal free—Recombinantly produced, free from exogenous viral contamination, and no animal-derived materials are used in the production process.
• Quality Stability: Batch production ensures stable and continuous batch manufacturing; there are no differences between product batches, ensuring consistent quality.
• Adequate Capacity: Yeasen possesses fermenters ranging from 5L to 1500L, meeting the varying batch requirements of different clients at different stages.

Applications

• In the production process of fusion peptides and proteins, it is used to remove fusion proteins located at the N-terminus of proteins to remove unwanted fusion tags, ensuring the accurate N-terminal sequence of recombinant fusion proteins.
• In the field of biopharmaceuticals, the production and preparation of GLP-1 analog peptide drugs are commonly used.

Specifications

Molecular Weight: *Due to the glycosylation effect after expression in Pichia pastoris, the molecular weight of the target protein shown by SDS-PAGE is about 40 kDa.

Activity Definition：*The cleavage efficiency may vary depending on the substrate. Generally, smaller proteins tend to have higher cleavage efficiency. The enzyme activity defined by Yeasen is based on a large protein with a molecular weight of approximately 64.6 kDa. For oligopeptide substrates, it can generally reach a maximum of 1 unit to cleave 500 μg.

Components

Storage 

The product can be stored at -25 ~ -15℃ for one year.

Figure

1. Enterokinase activity test

Figure 1. The enzyme cutting effect of Yeasen enterokinase (Cat#20395ES) is consistent with Supplier N*

[Note]: The 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.

2. 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).

3. Enterokinase cutting specificity test

Figure 3. Yeasen enterokinase (20395ES) has less non-specific cutting than Supplier N*

[Note]: Under the same conditions, Yeasen enterokinase (20395ES) and supplier N's enterokinase were used for enzyme cutting tests. The results show that after purification, the non-specific cutting impurities produced by Yeasen enterokinase (20395ES) are significantly lower than those of Supplier N*.

4. 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.

Instructions (Application Example)

1.  Reaction System:

2.  Reaction conditions: Mix well, react at 25°C for 16 hours.

Note: Recombinant enterokinase can be diluted with 25 mM Tris-HCl pH 8.0 to a solution containing 0.1 U per 1  μL for use.

Notes

1. This enzyme is highly potent and its activity is related to the sequence or structure of the protein to be cut. It is recommended to first do a gradient of protein input during testing, and to use the most appropriate amount.

2. The following conditions may have a consistent effect on the activity of rEK:

1) High ionic strength will inhibit its activity. Under 0.25 M NaCl conditions, it reduces the original activity of rEK by about 25%; under 2 M NaCl conditions, rEK is almost completely inhibited.

2) Reagents that inhibit its activity may include, >2 M Urea, >20 mM b-ME, >0.1% SDS, >50 mM imidazole, etc.

3) Conditions of pH<6 or pH>9 will also inhibit its activity.

3. Please wear the necessary PPE, such lab coat and gloves, to ensure your health and safety！

4. For research use only!

Related reading:

Unlocking the Power of Enterokinase

Documents:

Safety Data Sheet

20395_MSDS_HB250609.pdf

Manuals 

20395_Manuals_Ver. HB250609.pdf