肠激酶可以切割含有四个天冬氨酸的赖氨酸羧基端肽键：天冬氨酸 - 天冬氨酸 - 天冬氨酸 - 天冬氨酸 - 赖氨酸（DDDDK）。
氨基酸序列：此产品是一种高纯度的重组牛肠激酶轻链片段，氨基酸序列与牛肠激酶轻链一致。
酶学性质：肠激酶可去除位于蛋白质N-末端的融合蛋白，以除去不需要的融合标签。
基本特性：该酶经HPLC纯化，纯度高，特异性高，不含其他蛋白酶，可以在较宽pH范围（4.5-9.5）和较宽温度范围内有效切割融合蛋白。
使用方法
反应体系：
融合蛋白：0.1–1 mg/mL（蛋白总量 50–100 μg）
重组肠激酶：0.1–0.2 U
缓冲体系：25 mM Tris-HCl, pH 8.0
反应条件：25℃过夜酶切，或完全酶切需16-24h，用SDS-PAGE检测酶切效果。
结果验证：使用0.1 U本品酶切50 ug融合蛋白底物，反应温度是25℃，酶切不同时间梯度（8 h,16 h,24 h,40 h,48 h,64 h）
4℃低温酶切条件：4℃能对底物进行有效酶切，但需要将酶切时间延长至 48-64h，或增加 2-3 倍酶量。
酶切结果验证：使用0.1 U本品酶切50 ug融合蛋白底物，反应温度是4℃，酶切不同时间梯度（8 h,16 h,24 h,40 h,48 h,64 h）
酶切优化和放大：
优化：可以对酶切条件进行优化，例如缓冲液 pH，融合蛋白浓度，EK的量，以及酶切时间，使融合蛋白能在稳定条件下被酶切，用 SDS-PAGE 检测酶切效果。
放大：选择优化后的反应条件，按该条件等比例放大，用 SDS-PAGE 检测酶切效果。
去除重组肠激酶的方法：使用胰蛋白酶抑制剂亲和层析去除重组肠激酶。
产品优势
特异性：特定的蛋白酶，切割前面含有四个天冬氨酸的赖氨酸羧基端位点：天冬氨酸-天冬氨酸-天冬氨酸-天冬氨酸-赖氨酸(DDDDK)。
无动物源性：重组生产，无外源性的病毒污染，生产过程不使用任何动物源原料。
质量稳定：批量生产，可保证稳定连续的批次生产；产品批次间无差异，质量稳定。
纯度高：不含其他蛋白酶，无非特异性切割。
符合法规要求：生产设备和生产环境符合相关法规要求，生产过程完全遵循NSF ISO 9001:2015质量体系并符合GMP指导原则。
质量文件完整：按客户需求，可提供相关法规支持文件。
注意事项
酶切温度：不建议37℃条件下酶切，可能会有非特异性酶切出现。
酶切条件：在＞200mM 咪唑，或＞200mMNaCl，或＞5%甘油条件下，酶切效果受影响，可参照以下推荐方法进行酶切：
推荐方法：为获得理想酶切结果，请将样品透析到25mM Tris-HCl 8.0缓冲液中，再进行酶切。若不便透析，可将样品稀释到咪唑含量在100mM以下，NaCl浓度在50mM以下，甘油浓度小于5%以下进行酶切，酶的用量与蛋白的比例不变（即1U酶切500µg蛋白）。
如果样品溶液中含有上述成分中的一种或多种，且不便去除，此时适当增加酶量或延长酶切时间，也可达到较好酶切效果。
磷酸盐的影响：磷酸盐对Enterokinase有很强的抑制作用，痕量的磷酸盐都会严重影响Enterokinase的活性，因此在酶切体系内不能存在磷酸盐。
去除肠激酶：本品是具有高酶活力重组肠激酶，切割蛋白使用量少，可不考虑除去。后续如需去除重组肠激酶，可用阴离子交换树脂（eg.DEAE-FF）对其进行洗脱。推荐洗脱条件如下：
平衡缓冲液：25 mM Tris-HCl pH 8.0
洗脱缓冲液：25 mM Tris-HCl pH 8.0，含100 mM NaCl
酶切效果：蛋白酶切效果不好，可适当增加酶量，或适当延长酶切时间。
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
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