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

具有高底物特异性和高效率的蛋白酶是有限的标记融合蛋白消化所需要的。在这个项目中，我们研究了肝细胞生长因子激活剂（HGFA）的使用。首先，我们将hgfa -底物肽序列引入GST融合蛋白中。编码HGFA裂解序列的寡核苷酸（AKTKQLRVVNG）插入pGEX4T-3载体凝血酶裂解位点下游。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。表达的融合蛋白（GST-JIP1）用HGFA和凝血酶（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片段的广泛降解，而HGFA处理导致连接肽的切割而没有额外的消化。对于其他融合蛋白，GST-NK1（肝细胞生长因子的一种变体）和GST-Met激酶结构域，HGFA治疗是成功的。该系统在谷胱甘肽存在下工作，表明谷胱甘肽- sepharose的柱洗脱液可以不经预处理进行消化。然而，该系统在0.5-2M尿素的存在下不起作用，这表明尿素溶解蛋白在消化前应该是缺乏尿素的。

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.