Department of Chemistry, University of Southern California, Los Angeles, CA, United States.
Department of Chemistry, University of Southern California, Los Angeles, CA, United States; Department of Biological Sciences, University of Southern California, Los Angeles, CA, United States.
Methods Enzymol. 2022;675:63-82. doi: 10.1016/bs.mie.2022.07.004. Epub 2022 Aug 23.
A protein's structure and function often depend not only on its primary sequence, but also the presence or absence of any number of non-coded posttranslational modifications. Complicating their study is the fact that the physiological consequences of these modifications are context-, protein-, and site-dependent, and there exist no purely biological techniques to unambiguously study their effects. To this end, protein semisynthesis has become an invaluable chemical biology tool to specifically install non-coded or non-native moieties onto proteins in vitro using synthetic and/or recombinant polypeptides. Here, we describe two facets of protein semisynthesis (solid-phase peptide synthesis and expressed protein ligation) and their use in generating site-specifically glycosylated small heat shock proteins for functional studies. The procedures herein require limited specialized equipment, employ mild reaction conditions, and can be extended to myriad other proteins, modifications, and contexts.
蛋白质的结构和功能不仅取决于其一级序列，还取决于是否存在许多非编码的翻译后修饰。使这些修饰的研究变得复杂的是，这些修饰的生理后果取决于上下文、蛋白质和位点，并且没有纯粹的生物学技术可以明确研究它们的影响。为此，蛋白质半合成已成为一种非常宝贵的化学生物学工具，可使用合成和/或重组多肽在体外将非编码或非天然部分特异性地安装到蛋白质上。在这里，我们描述了蛋白质半合成（固相多肽合成和表达蛋白连接）的两个方面及其在生成用于功能研究的特异性糖基化小热休克蛋白中的应用。本文所述的程序需要有限的专用设备，采用温和的反应条件，并且可以扩展到许多其他蛋白质、修饰和情况。
