Enterokinase cleaves the peptide bond at the carboxyl terminal of lysine preceded by four aspartic acid residues: Asp-Asp-Asp-Asp-Lys (DDDDK).
Amino Acid Sequence: This product is high-purity recombinant bovine enterokinase light chain, whose amino acid sequence is identical to that of native bovine enterokinase light chain.
Enzymatic Properties: Enterokinase is capable of removing N-terminal fusion tags from target proteins.
Basic Characteristics: Purified by HPLC with high purity and strict substrate specificity, it is free of other proteases. It can efficiently cleave fusion proteins within a broad pH range (4.5-9.5) and wide temperature range.
Application Protocol
Reaction System
Fusion protein: 0.1–1 mg/mL (total protein: 50–100 μg)
Recombinant enterokinase: 0.1–0.2 U
Buffer: 25 mM Tris-HCl, pH 8.0
Reaction Conditions: Incubate overnight at 25 °C; complete cleavage takes 16–24 h. The cleavage efficiency is detected by SDS-PAGE.
Result Verification: 50 μg fusion protein substrate is digested with 0.1 U enterokinase at 25 °C for gradient durations: 8 h, 16 h, 24 h, 40 h, 48 h, 64 h.
Low-temperature Cleavage at 4 °C: Effective cleavage can be achieved at 4 °C, yet the reaction time needs to be extended to 48–64 h, or the enzyme dosage shall be increased by 2–3 folds.
Verification: Digest 50 μg fusion protein with 0.1 U enterokinase at 4 °C for 8 h, 16 h, 24 h, 40 h, 48 h and 64 h respectively.
Cleavage Optimization and Scale-up
Optimization: Adjust reaction parameters including buffer pH, fusion protein concentration, enterokinase dosage and incubation time to establish stable cleavage conditions, and verify results via SDS-PAGE.
Scale-up: Scale up the reaction system proportionally based on optimized conditions, and detect cleavage effect by SDS-PAGE.
Enterokinase Removal Method: Remove recombinant enterokinase by trypsin inhibitor affinity chromatography.
Product Advantages
High Specificity: Specifically recognizes and cleaves the carboxyl terminal of lysine in the sequence Asp-Asp-Asp-Asp-Lys (DDDDK).
Animal-origin Free: Produced via recombinant expression without animal-derived raw materials, free from exogenous viral contamination.
Stable Quality: Suitable for large-scale batch production with consistent performance among batches.
High Purity: No miscellaneous proteases and non-specific cleavage activity.
Regulatory Compliance: Production facilities and environment meet relevant regulations, complying with NSF ISO 9001:2015 quality management system and GMP guidelines.
Complete Documentation: Supporting regulatory documents are available upon request.
Precautions
Reaction Temperature: Cleavage at 37 °C is not recommended, as it may cause non-specific cleavage.
Inhibitory Substances: Cleavage activity is inhibited by over 200 mM imidazole, over 200 mM NaCl or more than 50% glycerol.
Recommended Operation: Dialyze samples into 25 mM Tris-HCl (pH 8.0) prior to digestion. If dialysis is unavailable, dilute samples to lower imidazole below 100 mM, NaCl below 50 mM and glycerol below 5% while keeping the enzyme-to-protein ratio unchanged (1 U enzyme for 500 μg protein).
If such inhibitors cannot be eliminated properly, increase enzyme dosage or prolong incubation time to improve cleavage efficiency.
Phosphate Inhibition: Phosphate strongly inhibits enterokinase activity; even trace amounts will severely reduce enzymatic activity. Phosphate is forbidden in the reaction system.
Enterokinase Elimination: This high-activity recombinant enterokinase is used in low dosage for protein cleavage, which can be retained without removal in most cases. If removal is required, use anion exchange resin (e.g. DEAE-FF) for elution:
Equilibration buffer: 25 mM Tris-HCl, pH 8.0
Elution buffer: 25 mM Tris-HCl (pH 8.0) containing 100 mM NaCl
Improved Cleavage Efficiency: Increase enzyme dosage or extend incubation time if unsatisfactory cleavage results are obtained.
F2605345