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Use of hepatocyte growth factor activator for limited digestion of tagged fusion proteins

使用肝细胞生长因子激活剂有限消化标记融合蛋白

基本信息

批准号：
11557181
负责人：
MIYAZAWA Keiji
金额：
$ 8.32万
依托单位：
TOKYO INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557181/
关键词：
protease fusion protein limited digestion recombinant protein

项目摘要

Proteases with high substrate specificity as well as high efficiency are desirable for limited digestion of tagged fusion proteins. In this project, we examined the use of hepatocyte growth factor activator (HGFA) for this purpose.First, we introduced an HGFA-substrate peptide sequence into GST fusion protein. Anoligonucleotide coding HGFA cleavage sequence (AKTKQLRVVNG) was inserted downstream of thrombin cleavage site of pGEX4T-3 vector. For assesing the utility of this system, JNK interacting protein-1 (JIP-1) was used as a model protein for expression and cleavage. The expressed fusion protein (GST-JIP1) was treated with HGFA as well as thrombin at substrate/enzyme ratio of 50/1 at 37℃ for 4 hours. Thrombin treatment caused extensive degradation of the JIP-1 moiety of the fusion protein, whereas HGFA treatment caused cleavage of the linker peptide without extra digestion. HGFA treatment was successful for other fusion proteins, GST-NK1 (a variant of hepatocyte growth factor) and GST-Met kinase domain. This system worked in the presence of glutathione, indicating that column eluate from glutathione-Sepharose can be processed for digestion without pretreatment. The system, however, failed to work in the presence of 0.5-2M urea, indicating that urea-solubilized proteins should be devoid of urea before digestion.

具有高底物特异性和高效率的蛋白酶对于标记融合蛋白的有限消化是理想的。在本项目中，我们研究了肝细胞生长因子激活剂 (HGFA) 的用途。首先，我们将 HGFA 底物肽序列引入 GST 融合蛋白中。将编码 HGFA 切割序列 (AKTKQLRVVNG) 的寡核苷酸插入 pGEX4T-3 载体凝血酶切割位点的下游。为了评估该系统的实用性，JNK 相互作用蛋白 1 (JIP-1) 被用作表达和切割的模型蛋白。将表达的融合蛋白（GST-JIP1）用HGFA以及凝血酶以底物/酶比为50/1在37℃下处理4小时。凝血酶处理导致融合蛋白的 JIP-1 部分广泛降解，而 HGFA 处理导致连接肽裂解而无需额外消化。 HGFA 治疗对其他融合蛋白、GST-NK1（肝细胞生长因子的变体）和 GST-Met 激酶结构域也取得了成功。该系统在谷胱甘肽存在的情况下工作，表明来自谷胱甘肽-琼脂糖凝胶的柱洗脱液可以在不进行预处理的情况下进行消化。然而，该系统在 0.5-2M 尿素存在的情况下无法工作，这表明尿素溶解的蛋白质在消化之前应该不含尿素。