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

对于标记融合蛋白的有限消化，具有高底物专一性和高效率的蛋白酶是可取的。在本项目中，我们研究了肝细胞生长因子激活剂(HGFA)的应用。首先，我们将HGFA-底物肽序列引入GST融合蛋白中。将编码HGFA裂解序列的寡核苷酸(AKTKQLRVVNG)插入pGEX4T-3载体凝血酶裂解位点的下游。为了验证该系统的实用性，以JNK相互作用蛋白-1(JIP-1)作为表达和切割的模型蛋白。表达的融合蛋白(GST-JIP1)在37℃的条件下，用重组人肝细胞生长因子和凝血酶在底物/酶比为50：1的条件下处理4h。凝血酶处理导致融合蛋白JIP-1部分的广泛降解，而HGFA处理导致连接肽的裂解而不需要额外的消化。HGFA对其他融合蛋白、GST-NK1(肝细胞生长因子的一种变体)和GST-Met激动域的治疗也是成功的。该体系在谷胱甘肽存在下工作，表明谷胱甘肽-琼脂糖柱洗脱液无需前处理即可消化。然而，该体系在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。

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.