Development of an effective method for purifying trypsin using a recombinant inhibitor

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-09-02 DOI:10.1016/j.pep.2024.106597

Chen Li , Zhaoxia Wang , Zejie Niu , Jiao Li , Lanxin Chen , Xiaodong Cui , Fang Li

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引用次数: 0

Abstract

A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and K_m value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30–50 °C and pH range of 4.0–10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.

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开发一种利用重组抑制剂纯化胰蛋白酶的有效方法。

通过在环氧氯丙烷活化的交联琼脂糖凝胶（Selfinose CL 6B）上共价固定荞麦胰蛋白酶抑制剂（BTI），制备了一种胰蛋白酶亲和材料。活化 Selfinose CL 6B 的最佳条件是 15%环氧氯丙烷和 0.8 M NaOH，温度为 40°C，时间为 2 小时；固定 BTI 的最佳 pH 值为 9.5。BTI-Sefinose CL 6B 的最大吸附容量为 2.25 毫克胰蛋白酶/（克支持物）。该材料还具有良好的重复使用性，在循环 30 次后仍能保持 90% 以上的初始吸附容量。使用 BTI-Selfinose CL 6B 通过一步亲和层析从蝗虫匀浆中获得了高纯度胰蛋白酶。以 N-苯甲酰基-DL-精氨酸-硝基苯胺为底物，测定了蝗虫胰蛋白酶的分子质量和 Km 值分别为 27 kDa 和 0.241 mM。胰蛋白酶活性的最佳温度和 pH 值分别为 55 °C 和 9.0。该酶在 30-50 °C 的温度范围和 4.0-10.0 的 pH 范围内表现出良好的稳定性。BTI-Selfinose CL 6B 在制备高纯度胰蛋白酶和从不同物种中发现更多新型胰蛋白酶方面具有潜在的应用价值。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.