Versatile protein biotinylation strategies for potential high-throughput proteomics
Versatile protein biotinylation strategies for potential high-throughput proteomics
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DOI：
10.1021/ja037914g
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发表时间：
2004-02-04
影响因子：
15
通讯作者：
Yao, SQ
中科院分区：
文献类型：
--
作者：
Lue, RYP;Chen, GYJ;Yao, SQ
我们提出了有效的蛋白位点特异性生物素化的内部介导方法。在体外或活细胞中，由辅助性蛋白剪接产生的活性c端硫酯是一个有吸引力的、唯一的附着含半胱氨酸生物素的位点。利用这些新的生物素化策略，我们能够以潜在的高通量、高含量的方式有效地将来自不同生物来源的许多蛋白质生物素化。其中一些蛋白质随后以非常简单的方式固定在不同的亲和素功能化的固体表面上，用于蛋白质微阵列和表面等离子体共振（SPR）光谱等应用，突出了使用生物素作为蛋白质纯化/固定方法的众多优势，而不是其他标签（例如，GST， His-tag等）。此外，我们的内部介导策略在许多方面比其他蛋白质生物素化策略提供了关键优势。我们还首次成功地证明了在细菌和哺乳动物活细胞内以及在无细胞蛋白质合成系统中，胰岛素介导的蛋白质生物素化都能充分进行。综上所述，我们的研究结果表明，这些内部介导的策略具有潜在的高通量蛋白质组学应用的多功能性。它们也可作为各种体外和体内蛋白质的生物化学和生物物理研究的有用工具。
We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/ immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo.