重组肠激酶

重组牛肠激酶

Enterokinase | EK | Enteropeptidase

R141087-0.5KU

0.5KU

无动物源

≥5 units/μl

重组牛肠激酶是一种高纯度的重组牛肠激酶轻链片段。该酶经高效液相色谱（HPLC）纯化，纯度高、特异性高，不含其他蛋白酶。肠激酶可切割含有四个天冬氨酸的赖氨酸羧基端肽键，具体序列为天冬氨酸 - 天冬氨酸 - 天冬氨酸 - 天冬氨酸 - 赖氨酸（DDDDK）。其适用范围广，能在较宽的 pH 范围（4.5-9.5）和较宽温度范围内有效切割融合蛋白，还可去除位于蛋白质 N - 末端的融合蛋白，从而除去不需要的融合标签。 细胞定位： Membrane; Single-pass type II membrane protein 序列相似性： Belongs to the peptidase S1 family. 推荐使用方法 1）25℃酶切条件 按照酶活性定义，酶切条件举例如下： 在 25mM Tris-HCl（pH8.0）体系中： 融合蛋白浓度为 0.1-1mg/ml（蛋白总量 50-100μg） 重组肠激酶（EK）用量为 0.1-0.2U 于 25℃过夜酶切，或需 16-24h 实现完全酶切，酶切效果可通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）检测。 2）4℃低温酶切条件 4℃环境下可对底物进行有效酶切，但需将酶切时间延长至 48-64h，或增加 2-3 倍酶量。 3）酶切优化和放大 优化：可对酶切条件进行优化，如缓冲液 pH、融合蛋白浓度、肠激酶（EK）用量及酶切时间等，确保融合蛋白在稳定条件下被酶切，酶切效果通过 SDS-PAGE 检测。 放大：选择优化后的反应条件，按该条件等比例放大酶切反应，酶切效果通过 SDS-PAGE 检测。 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。 注意： 不建议在 37℃条件下进行酶切，此温度下可能出现非特异性酶切。 储存和运输稳定性 酶液储存稳定性： -20℃条件下，12 个月内稳定；25℃条件下，一周内无活性损失。缓冲体系为 50mM Tris-HCl（pH8.0）、250mM NaCl、2mM Ca²⁺、50% 甘油。 酶液反复冻融稳定性： 反复冻融 5 次，酶无活性损失。 运输稳定性： 采用蓝冰保温运输，酶保持稳定。 常见影响肠激酶活性的因素 当体系中存在＞200mM 咪唑、或＞200mM NaCl、或＞5% 甘油时，酶切效果会受影响。可参照以下推荐方法进行酶切： 1） 为获得理想酶切结果，建议将样品透析到 25mM Tris-HCl（pH8.0）缓冲液中，再进行酶切。 2） 若不便透析，可将样品稀释，使咪唑含量在 100mM 以下、NaCl 浓度在 50mM 以下、甘油浓度小于 5%，之后进行酶切，酶的用量与蛋白的比例保持不变（即 1U 酶可切割 500μg 蛋白）。 3） 若样品溶液中含有上述影响成分中的一种或多种，且不便去除，此时可适当增加酶量或延长酶切时间，同样可达到较好的酶切效果。 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.

Responsible for initiating activation of pancreatic proteolytic proenzymes (trypsin, chymotrypsin and carboxypeptidase A). It catalyzes the conversion of trypsinogen to trypsin which in turn activates other proenzymes including chymotrypsinogen, procarboxypeptidases, and proelastases.

≥ 5.0 U/µl

重组表达

25.8 ±2.6kDa

No tag

E. coli Accession #: P98073,P98072 | E. coli

牛(Bovine)

Yes

Yes