Sortase A-Catalyzed Site-Specific Coimmobilization on Microparticles via Streptavidin
Sortase A-Catalyzed Site-Specific Coimmobilization on Microparticles via Streptavidin
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DOI：
10.1021/la2047933
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发表时间：
2012-02-21
期刊：
影响因子：
3.9
通讯作者：
Kondo, Akihiko
中科院分区：
文献类型：
--
作者：
Matsumoto, Takuya;Tanaka, Tsutomu;Kondo, Akihiko
通过结合链霉亲和素生物素亲和性和分选酶A（SrtA）催化的转肽作用的独特方法设计微粒表面。使用链霉亲和素生物素亲和性将亮氨酸-脯氨酸-谷氨酸-苏氨酸-甘氨酸标记的链霉亲和素（Stav-LPETG）固定在表面上，并将GGGGG标记的红色荧光蛋白（Gly 5-RFP）与SrtA缀合。然后将生物素化的异硫氰酸荧光素（生物素FITC）与Stav-LPETG中的残留生物素结合位点结合。所得颗粒具有通过Stav-LPETG固定在表面上的RFP和FITC，并且使用荧光显微镜观察RFP和FITC相关的荧光。最后，GGG标记的葡萄糖氧化酶和生物素化辣根过氧化物酶被固定在微粒表面上，产生能够检测葡萄糖的功能性颗粒。该颗粒可以重复使用，并且在检测葡萄糖方面比使用化学修饰制备的颗粒更灵敏。我们的方法提供了一个简单的策略，位点特异性的分子表面上的共固定，并扩大了蛋白质杂交装置的使用。
A microparticle surface was designed by the unique method incorporating streptavidin biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG) was immobilized on the surface using streptavidin biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-associated fluorescence was observed using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepared using chemical modification. Our method provides a simple strategy for site-specific coimmobilization on molecular surfaces and expands the use of protein hybrid devices.