Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.
J Biotechnol. 2011 Mar 10;152(1-2):37-42. doi: 10.1016/j.jbiotec.2011.01.008. Epub 2011 Jan 22.
Streptavidin is tetrameric protein which has tight and specific biotin binding affinity, and streptavidin modification of proteins or small molecules is widely used for biotechnology tool. Here, we demonstrate site-specific streptavidin-protein conjugation using enzymes. We focused on sortase A, a transpeptidase from Staphylococcus aureus. A streptavidin-tagged LPETG motif (Stav-LPETG) was expressed in Escherichia coli. We achieved soluble streptavidin expression in E. coli without refolding using a cold shock expression system. Then we successfully conjugated Stav-LPETG with pentaglycine-appended green fluorescence protein (Gly5-GFP) or triglycine-appended glucose oxidase (Gly3-GOD) using sortase A. SDS-PAGE analysis showed site-specific tetrameric streptavidin-protein conjugation with the tagged proteins. In addition, the functions of a Stav-GOD conjugate, i.e., biotin-binding and glucose oxidase activity, were significantly higher compared to those of streptavidin-GOD conjugates prepared by chemical modification.
链霉亲和素是一种四聚体蛋白，具有紧密和特异性的生物素结合亲和力，链霉亲和素修饰的蛋白质或小分子被广泛用于生物技术工具。在这里，我们使用酶展示了定点链霉亲和素-蛋白质偶联。我们专注于来自金黄色葡萄球菌的转肽酶即 sortase A。在大肠杆菌中表达了带有链霉亲和素标签的 LPETG 基序（Stav-LPETG）。我们使用冷休克表达系统实现了无需复性的可溶性链霉亲和素表达。然后，我们使用 sortase A 成功地将 Stav-LPETG 与五聚甘氨酸缀合的绿色荧光蛋白（Gly5-GFP）或三甘氨酸缀合的葡萄糖氧化酶（Gly3-GOD）偶联。SDS-PAGE 分析表明，带有标签的蛋白质的定点四聚体链霉亲和素-蛋白质偶联。此外，与通过化学修饰制备的链霉亲和素-GOD 缀合物相比，Stav-GOD 缀合物的生物素结合和葡萄糖氧化酶活性显著更高。
