| Field | Specification |
|---|---|
| Mfr No | |
| Product Type | |
| Shipping | |
| Source | Recombinant (E. coli) |
| Storage |
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
|
Source |
Yeast recombinant expression |
|
Molecular Weight* |
Theoretical value 22.7 kDa |
|
Appearance |
Sterile liquid |
|
Storage Buffer |
50 mM Tris-HCl, 250 mM NaCl, 2 mM CaCl2, 50% Glycerol, pH 8.0 |
|
Enzyme Concentration |
5 U/μL |
|
Purity |
≥95% |
|
Activity Definition* |
One unit of activity is defined as the amount of enzyme required to cut 95% of the 50 μg fusion protein with enterokinase cleavage site (Enterokinase Positive Substrate, molecular weight approximately 64.6 kDa, Cat#20391ES) under the condition of 25°C, 12-16 hours, in a buffer system (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 8.0). |
Glycerol Content |
Contains Glycerol |
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
|
Components No. |
Name |
20395ES60 |
20395ES76 |
20395ES90 |
20395ES92 |
20395ES94 |
|
20395 |
Recombinant Enterokinase, His Tag |
500 U |
5000 U |
100 KU |
1 MU （1000 KU） |
Storage
The product can be stored at -25 ~ -15℃ for 2 years.
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:
|
Fusion protein |
50 μg |
|
Enterokinase |
0.1-1 U (adjustable according to actual conditions) |
|
Reaction Buffer （20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 8.0） |
Up to 50-500 μL(protein concentration 0.1-1 mg/mL) |
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:
Store this enzyme at -20°C and avoid repeated freeze-thaw cycles to preserve catalytic activity. The product is shipped Dry Ice and remains stable for up to one year from the date of manufacture when stored under recommended conditions. Aliquoting the stock solution into single-use volumes is recommended for enzymes used infrequently to minimize thermal cycling of the bulk stock.
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 recombit fusion proteins. In the field of biopharmaceuticals, the production and preparation of GLP-1 analog peptide drugs are commonly used.. Always verify compatibility with your specific template, buffer, and downstream workflow.
One unit (U/mg) is defined as the amount of protease activity per milligram of enzyme protein under standardized assay conditions (substrate concentration, pH, temperature, and incubation time).
This enzyme is produced as Recombinant (E. coli) and supplied as a Research Use Only (RUO) reagent. Each lot is subjected to activity assay, purity assessment by SDS-PAGE, and functional validation prior to release. A Certificate of Analysis (CoA) and Safety Data Sheet (SDS) are available on request.
Most serine proteases (e.g., Proteinase K) are inhibited by PMSF (1 mM) and heat-inactivated at 95°C for 10 min. Proteinase K retains activity across a broad pH range (4–12) and tolerates SDS, urea, and other denaturants, making it particularly effective for nucleic acid extraction from challenging samples including FFPE tissue.
Yeasen Biotechnology supports custom enzyme solutions across multiple service lines — from GMP-grade bulk supply to directed enzyme engineering. Contact BioHippo to discuss requirements and initiate a project inquiry.
▶ GMP-Grade & Bulk Supply
Select Yeasen enzymes are available in GMP grade, manufactured in an ISO 13485-certified UCF.ME™ ultra-clean molecular enzyme facility with FDA Drug Master File (DMF) support.
- GMP-grade release testing and CoA documentation
- ISO 13485-certified production facility
- Scalable from milligram to multi-gram quantities
- Consistent lot-to-lot activity specifications
▶ Glycerol-Free & Custom Formulation
Glycerol-free enzyme formats are available for applications requiring lyophilization compatibility, liquid handling automation, or direct IVD master mix integration.
- Glycerol-free liquid format (standard and custom buffers)
- Lyophilization-ready enzyme preparation
- Custom reaction buffer optimization for specific assay conditions
- Compatible with freeze-drying workflows for point-of-care formats
▶ Molecular IVD RDC Service
Yeasen's Research and Development Contracting (RDC) team delivers end-to-end solutions for molecular diagnostic product development, covering enzyme selection through clinical validation support.
- Enzyme selection and performance matching
- Primer/probe design and reaction buffer optimization
- Sensitivity, specificity, and precision validation studies
- Stability studies and SNP evaluation
- Instrument platform compatibility assessment
▶ ZymeEditor™ Enzyme Engineering
Yeasen's proprietary ZymeEditor™ directed evolution and rational design platform enables the development of custom enzyme variants with tailored performance characteristics not available in off-the-shelf products.
- Directed evolution for enhanced thermostability, processivity, or fidelity
- Rational design for altered substrate specificity or cofactor requirements
- Library screening from Yeasen's proprietary enzyme variant collection
- Scale-up to commercial quantities upon candidate confirmation
ⓘ Customization services are fulfilled by Yeasen Biotechnology. Lead times and minimum order quantities vary by service type. Contact BioHippo for project scoping and pricing.
