Ejendal Karin F K, Fraseur Julia G, Kinzer-Ursem Tamara L
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA.
Methods Mol Biol. 2019;2033:149-165. doi: 10.1007/978-1-4939-9654-4_11.
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.
在蛋白质表达系统中能够进行共翻译蛋白质标记的方法已在健康、工程和医学领域有着广泛的应用。生物正交化学能够在无需事先富集或纯化的情况下，使活细胞中感兴趣的蛋白质或生物分子与底物（荧光团、金纳米颗粒、聚合物等）共轭，同样推动了研究和改造细胞及生物分子系统的技术进步。在这两者的交叉领域，在特定感兴趣位点对蛋白质进行化学酶法标记，并随后在无需事先纯化的情况下将其与底物进行选择性生物共轭，极大地简化了工作流程，使得蛋白质在共轭之前能够尽可能长时间地保留在天然表达量中，在共轭后易于分离，并在共轭后保持功能活性。在此，我们展示了用肉豆蔻酸的叠氮衍生物（一种小的、可溶的、含14个碳的脂肪酸）在N端表达和标记感兴趣蛋白质的方法及方案，并将标记的蛋白质与荧光团和金纳米颗粒底物共轭。这些方法可以扩展到用其他肉豆蔻酰衍生物标记蛋白质，并与其他感兴趣的固体或聚合物底物共轭。
