Recombinant bovine enteropeptidase is a high-purity recombinant fragment of the light chain of bovine enteropeptidase. Purified by High-Performance Liquid Chromatography (HPLC), this enzyme exhibits high purity and specificity, with no other proteases present. It can cleave the peptide bond at the carboxyl terminus of lysine that is preceded by four aspartic acid residues, with the specific sequence being Aspartic Acid-Aspartic Acid-Aspartic Acid-Aspartic Acid-Lysine (DDDDK). Boasting a wide range of applications, it can effectively cleave fusion proteins within a broad pH range (4.5-9.5) and a wide temperature range. Additionally, it can remove fusion proteins located at the N-terminus of the target protein, thereby eliminating unwanted fusion tags.
Recommended Usage Methods
1) Enzymatic Cleavage Conditions at 25°C
In accordance with the definition of enzyme activity, an example of the enzymatic cleavage conditions is as follows:
In a 25mM Tris-HCl (pH 8.0) system:
The concentration of the fusion protein is 0.1-1mg/ml (with a total protein amount of 50-100μg).
The dosage of recombinant enteropeptidase (EK) is 0.1-0.2U.
Perform enzymatic cleavage at 25°C overnight, or allow 16-24 hours to achieve complete cleavage. The cleavage effect can be detected by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).
2) Low-Temperature Enzymatic Cleavage Conditions at 4°C
Effective enzymatic cleavage of the substrate can be achieved at 4°C, but it is necessary to extend the cleavage time to 48-64 hours or increase the enzyme dosage by 2-3 times.
3) Optimization and Scaling-Up of Enzymatic Cleavage
Optimization: The enzymatic cleavage conditions can be optimized, such as the pH of the buffer, the concentration of the fusion protein, the dosage of enteropeptidase (EK), and the cleavage time. This ensures that the fusion protein is cleaved under stable conditions, and the cleavage effect is detected by SDS-PAGE.
Scaling-Up: Select the optimized reaction conditions and scale up the enzymatic cleavage reaction proportionally based on these conditions. The cleavage effect is detected by SDS-PAGE.
Method for Removing Recombinant Enteropeptidase
Recombinant enteropeptidase can be removed using trypsin inhibitor affinity chromatography.
Note: Enzymatic cleavage at 37°C is not recommended, as non-specific cleavage may occur at this temperature.
Storage and Transportation Stability
Storage Stability of Enzyme Solution: It remains stable for 12 months at -20°C; no activity loss occurs within one week at 25°C. The buffer system consists of 50mM Tris-HCl (pH 8.0), 250mM NaCl, 2mM Ca²⁺, and 50% glycerol.
Stability of Enzyme Solution During Repeated Freezing and Thawing: No activity loss of the enzyme is observed after 5 repeated freeze-thaw cycles.
Transportation Stability: The enzyme remains stable when transported with blue ice insulation.
Common Factors Affecting Enteropeptidase Activity
When the system contains >200mM imidazole, or >200mM NaCl, or >5% glycerol, the enzymatic cleavage effect will be affected. The following recommended methods can be referred to for enzymatic cleavage:
1) To achieve an ideal cleavage effect, it is advisable to dialyze the sample into a 25mM Tris-HCl (pH 8.0) buffer before performing enzymatic cleavage.
2) If dialysis is not convenient, the sample can be diluted to ensure the imidazole content is below 100mM, the NaCl concentration is below 50mM, and the glycerol concentration is less than 5% before enzymatic cleavage. The ratio of enzyme dosage to protein amount remains unchanged (i.e., 1U of enzyme can cleave 500μg of protein).
3) If the sample solution contains one or more of the aforementioned interfering components and their removal is not feasible, appropriate increases in enzyme dosage or extensions of cleavage time can also result in a good enzymatic cleavage effect.
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