Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.
Langmuir. 2012 Feb 21;28(7):3553-7. doi: 10.1021/la2047933. Epub 2012 Feb 7.
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.
采用链亲和素-生物素亲和和转肽酶 SrtA 催化的转肽作用的独特方法设计了微粒表面。通过链亲和素-生物素亲和将带有亮氨酸-脯氨酸-谷氨酸-苏氨酸-甘氨酸标签的链霉亲和素（Stav-LPETG）固定在表面上，并将带有 GGGGG 标签的红色荧光蛋白（Gly5-RFP）与 SrtA 缀合。然后将生物素化的异硫氰酸荧光素（biotin-FITC）结合到 Stav-LPETG 中的残留生物素结合位点上。由此得到的颗粒通过 Stav-LPETG 将 RFP 和 FITC 固定在表面上，并通过荧光显微镜观察 RFP 和 FITC 相关的荧光。最后，将葡萄糖氧化酶的 GGG 标签和辣根过氧化物酶的生物素化修饰固定在微粒子表面上，得到能够检测葡萄糖的功能颗粒。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。我们的方法为分子表面的特异性共固定提供了一种简单的策略，并扩展了蛋白质杂合器件的用途。
