Site-specific biotinylation of purified proteins using BirA.
Site-specific biotinylation of purified proteins using BirA.
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DOI：
10.1007/978-1-4939-2272-7_12
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发表时间：
2015
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影响因子：
--
通讯作者：
Howarth, Mark
中科院分区：
文献类型：
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作者：
Fairhead, Michael;Howarth, Mark
生物素与链亲和素或亲和素之间的结合是已知最强的非共价生物相互作用之一。数十年来，亲和素-生物素相互作用在生物研究和生物技术中得到了广泛的应用。因此，用生物素标记纯化的蛋白质是实现蛋白质捕获、固定化和功能化以及多聚化或桥接分子的有力途径。化学生物素化通常会产生异质产物，这可能会损害功能。利用大肠杆菌生物素连接酶（BirA）进行的酶促生物素化反应具有高度特异性，可将生物素共价结合到含有15个氨基酸的AviTag肽上，从而获得高产量的均匀产物。AviTag可以方便地添加到靶蛋白的n端、c端或暴露环中。本文描述了通过反相PCR插入AviTag、从大肠杆菌中纯化与谷胱甘肽-s转移酶（GST-BirA）融合的BirA、纯化蛋白的BirA生物素化以及通过SDS-PAGE进行凝胶移位分析以量化生物素化程度的过程。
The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.