Department of Biological Sciences, National University of Singapore, Singapore.
Curr Med Chem. 2009;16(34):4527-43. doi: 10.2174/092986709789760706.
Methods that allow visualisation of proteins in living systems, in real time have been key to our understanding of the molecular underpinnings of life. Although the use of genetically encoded fusions to fluorescent proteins have greatly advanced such studies, the large size of these tags and their ability to perturb protein activity has been major limitations. Attempts to circumvent these issues have seen the genesis of complementary strategies to chemically label/modify proteins. Thus, chemical labelling approaches seek to "decorate" biomolecules in live cells through the site-specific introduction of a small, non-native chemical tag (or reporter group). The introduced tag is minimally invasive such that the activity and/or function of the target molecule in not perturbed/compromised by its inclusion. In most cases, this modification is brought about by fusing target biomolecules to protein domains/peptide tags or via the incorporation of reactive "handles" by either exploiting the cell's biosynthetic machinery or during protein synthesis. Selective tagging of the biomolecule then proceeds via a bioorthogonal chemical reaction following exogenous addition of probe(s). Depending on the nature of the probe, the method can be applied to either visualise/track the dynamics of target molecule(s) in their native cellular milieu or for affinity enrichment for further downstream applications. The versatility of these approaches has been demonstrated by their ability to tag not just proteins but also intractable biomolecules like lipids and glycans. In this review, we summarise the various strategies available to "chemically" tag proteins and provide a comparative analysis their advantages and disadvantages. We also highlight the many creative applications of such methodologies and discuss their future prospects.
方法使蛋白质在活细胞中的可视化，实时已经成为我们对生命分子基础理解的关键。尽管使用遗传编码融合到荧光蛋白极大地推进了这些研究，但这些标签的体积大及其对蛋白质活性的干扰能力是主要的局限性。为了规避这些问题，人们尝试了用化学标记/修饰蛋白质的互补策略。因此，化学标记方法试图通过在活细胞中特异性地引入一个小的、非天然的化学标记（或报告基团）来“修饰”生物分子。引入的标记的侵入性很小，不会干扰/损害目标分子的活性和/或功能。在大多数情况下，这种修饰是通过将靶生物分子融合到蛋白质结构域/肽标签上，或者通过利用细胞的生物合成机制或在蛋白质合成过程中引入反应性“手柄”来实现的。然后，通过在外源性添加探针后进行生物正交化学反应来选择性地标记生物分子。根据探针的性质，该方法可用于可视化/跟踪目标分子在其天然细胞环境中的动力学，或用于亲和富集，以用于进一步的下游应用。这些方法的多功能性已经通过它们不仅能够标记蛋白质，还能够标记难以处理的生物分子（如脂质和聚糖）得到了证明。在这篇综述中，我们总结了各种“化学”标记蛋白质的策略，并对它们的优缺点进行了比较分析。我们还强调了这些方法的许多创造性应用，并讨论了它们的未来前景。
