YaxinBio Enterokinase is a kind of highly purified recombinant bovine enterokinase light chain. The enzyme has been extensively purified by HPLC and there are no traces of other contaminating proteases. Enterokinase specifically hydrolyzes peptide bond at the carboxyl side of lysine residue preceded by four aspartic acids: Asp-Asp-Asp-Asp-Lys (DDDDK). So, enterokinase can remove N-terminal fusion protein or tags to get aim protein with native amino acids sequence.
|Source||Recombinant E. coli|
|Appearance||Clear,colorless to yellowish liquid|
|Purity(SDS-PAGE)||Single major band|
|Molecular Weight(SDS-PAGE)||25.8 ±2.6 kDa|
UNIT DEFINITION：One unit is defined as the amount of enzyme needed to cleave 0.5mg of fusion protein in 12 to16 hours to get 95% completion at 25°C in 25mMTris-HCl, pH8.0. Substrate: a special fusion protein.
3.RECOMMEND USAGE CONDITION:
1) Cutting condition: 25mM Tris-HCl 8.0
Fusion protein concentration: 0.1-1mg/ml (total protein content: 50-100ug)
EK content: 0.1-0.2U
Time: overnight or 16h-24h for digestion.
If digestion under 2-8°C,prolong the digestion time two folds,or more enzyme added to short the digestion time.
2) 4°C low temperature digestion conditions:
4°C can effectively digest the substrate, but need to extend the digestion time to 48-64h, or increase the amount of 2-3 times the amount of enzyme.
3) Enzyme cleavage optimization and amplification
Optimization: Enzyme cleavage conditions can be optimized, such as buffer pH, fusion protein concentration, EK amount, and digestion time, so that the fusion protein can be digested under stable conditions, SDS-PAGE detection of digestion effect.
Amplification: Select the optimized reaction conditions, according to the conditions such as the proportion of amplification, SDS-PAGE detection of digestion effect.
4) Method for removing recombinant enterokinase: The recombinant enterokinase was removed using trypsin inhibitor affinity chromatography (e.g., sigma T0637).
5) Note: do not recommend cutting at 37°C conditions, there may be non-specific enzyme digestion.
4.COMMON COMPONENTS INFLUENCE THE ACTION OF ENTEROKINASE
＞200mM imidazole or ＞200mM NaCl or ＞5% glycerin, the reaction may be effected.The following suggestions are given:
1) To receive the optimum result, please dialyze the sample to 25 mMTris-HCl, pH 8.0.
2) If the dialysis is inconvenient, please dilute the sample to ＜100mM imidazole, ＜50mMNaCl, ＜5% glycerin, and the proportion of fusion protein and EK may not be changed (1U:0.5mg fusion protein).
3) If there are one or more components in samples, and cannot be removed, suggest to increase the content of EK in reaction system or extend the reaction time.
5. STABILITY OF STORAGE AND TRANSPORT
Stability of storage: Recombinant enterokinase should be stored under -20°C in sealed container. It is stable within 24 months.If stored at 25°C ,it is stable within one week.It is no activity loss after 5 times repeated freezing and thawing.
Stability of transport: The product is stable by blue ice insulation transport.
Animal origin free: Recombinant enterokinase is no exogenous virus contamination,and any animal origin material is not used in the production process.
Stable quality: Mass production can ensure stable and continuous batch production.It is no difference between the batch and the product quality is stable.
High purity: Higher specific activity.Host protein residues is less than the limits of biological products.
Lyophilized powder: The product is lyophilized powder and is easy to store and transport.
Compliance with regulatory requirements: Production equipment and production environment comply with relevant regulatory requirements, and the production process is in full compliance with NSF ISO 9001: 2008 quality system and GMP guidelines.
Complete quality documents: we can provide relevant regular support files in according to customers’ requirement.
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