Protein Labeling and Bioconjugation Using N-Myristoyltransferase
Protein Labeling and Bioconjugation Using N-Myristoyltransferase
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DOI：
10.1007/978-1-4939-9654-4_11
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发表时间：
2019-01-01
期刊：
影响因子：
--
通讯作者：
Kinzer-Ursem, Tamara L.
中科院分区：
文献类型：
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作者：
Ejendal, Karin F. K.;Fraseur, Julia G.;Kinzer-Ursem, Tamara L.
允许在蛋白质表达系统内共翻译标记蛋白质的方法在健康、工程和医学领域具有广泛的应用。生物正交化学允许将感兴趣的蛋白质或生物分子与活细胞中的底物（荧光团、金纳米颗粒、聚合物等）缀合，而无需事先富集或纯化，这同样使得研究和工程细胞和生物分子系统的技术取得了进步。在这些交叉点上，在特定感兴趣位点对蛋白质进行化学酶标记，以及随后无需事先纯化而选择性生物缀合到底物上，极大地简化了工作流程，使蛋白质在缀合前尽可能长时间地驻留在天然表达体积中，在缀合后易于分离，并在缀合后保持功能活性。在这里，我们提出了用肉豆蔻酸（一种小的可溶性 14 碳脂肪酸）叠氮衍生物在 N 末端表达和标记感兴趣的蛋白质的方法和方案，并将标记的蛋白质与荧光团和金纳米颗粒底物缀合。这些方法可以扩展到用其他肉豆蔻酰衍生物标记蛋白质以及与其他感兴趣的固体或聚合物底物缀合。
Methods that allow for labeling of proteins cotranslationally within protein expression systems have had wide-ranging applications in health, engineering, and medicine. Bioorthogonal chemistries that allow for conjugation of proteins or biomolecules of interest to substrates (fluorophores, gold nanoparticles, polymers, etc.) in living cells without prior enrichment or purification have likewise enabled advances in technology to study and engineer cellular and biomolecular systems. At the intersection of these, chemoenzymatic labeling of proteins at specific sites of interest and their subsequent selective bioconjugation to substrates without prior purification has dramatically streamlined workflows that allow proteins to reside in the native expression volumes as long as possible prior to conjugation, be readily isolated upon conjugation, and remain functionally active after conjugation. Here we present methods and protocols to express and label proteins of interest at the N-terminus with azide derivatives of myristic acid, a small, soluble, 14-carbon fatty acid, and conjugate the labeled protein to fluorophores and gold nanoparticle substrates. These methods can be extended to label proteins with other myristoyl derivatives and to conjugation to other solid or polymeric substrates of interest.