Site-specific tetrameric streptavidin-protein conjugation using sortase A
Site-specific tetrameric streptavidin-protein conjugation using sortase A
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DOI：
10.1016/j.jbiotec.2011.01.008
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发表时间：
2011-03-10
影响因子：
4.1
通讯作者：
Kondo, Akihiko
中科院分区：
文献类型：
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作者：
Matsumoto, Takuya;Sawamoto, Shiori;Kondo, Akihiko
链霉亲和素是一种四聚体蛋白，具有紧密而特异的生物素结合亲和力，对蛋白质或小分子进行链霉亲和素修饰被广泛应用于生物技术工具。在这里，我们展示了使用酶的位点特异性链霉亲和素-蛋白质缀合。我们重点关注分选酶A，这是一种来自金黄色葡萄球菌的转肽酶。在大肠杆菌中表达链霉亲和素标记的LPETG基序（Stav-LPETG）。我们在大肠杆菌中实现了可溶性链霉亲和素的表达。大肠杆菌中，而不使用冷休克表达系统进行重折叠。然后，我们成功地将Stav-LPETG与附加有五甘氨酸的绿色荧光蛋白（Gly 5-GFP）或附加有三甘氨酸的葡萄糖氧化酶（Gly 3-GOD）使用分选酶A缀合。SDS-PAGE分析显示位点特异性四聚体链霉亲和素-蛋白质与标记的蛋白质缀合。此外，Stav-GOD共轭的功能，即，生物素结合和葡萄糖氧化酶活性显著高于通过化学修饰制备的链霉亲和素-GOD缀合物。(C)2011 Elsevier B. V.保留所有权利。
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. (C) 2011 Elsevier B.V. All rights reserved.