Department of Chemistry, University of Southern California, Los Angeles, CA, USA.
Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA.
Methods Mol Biol. 2020;2133:313-326. doi: 10.1007/978-1-0716-0434-2_15.
α-Synuclein is a small aggregation-prone protein associated with Parkinson's disease (PD). The protein's biochemical and biophysical properties can be heavily influenced by various types of posttranslational modification (PTMs) such as phosphorylation, ubiquitination, and glycosylation. To understand the site-specific effects of various PTMs have on the protein and its aggregation, obtaining a homogeneous sample of the protein of interest with the specific modification of interest is key. Expressed protein ligation (EPL) has emerged as robust tool for building synthetic proteins bearing site-specific modifications. Here, we outline our approach for building α-synuclein with site specific O-GlcNAc modifications, an intracellular subtype of glycosylation that has been linked to the inhibition of protein aggregation. More specifically, we provide specific protocols for the synthesis of α-synuclein bearing an O-GlcNAc modification at threonine 72, termed α-synuclein(gT72). However, this general approach utilizing two recombinant fragments and one synthetic peptide is applicable to other sites and types of modifications and should be transferable to various other protein targets, including aggregation prone proteins like tau and TDP-43.
α-突触核蛋白是一种与帕金森病（PD）相关的小聚集倾向蛋白。该蛋白的生化和生物物理特性可能会受到各种类型的翻译后修饰（PTMs）的严重影响，如磷酸化、泛素化和糖基化。为了了解各种 PTM 对蛋白质及其聚集的特异性影响，获得具有感兴趣的特定修饰的感兴趣蛋白质的同质样品是关键。表达蛋白连接（EPL）已成为构建具有位点特异性修饰的合成蛋白的强大工具。在这里，我们概述了构建具有特定 O-GlcNAc 修饰的 α-突触核蛋白的方法，O-GlcNAc 修饰是一种与蛋白质聚集抑制有关的细胞内糖基化亚型。更具体地说，我们提供了在苏氨酸 72 位带有 O-GlcNAc 修饰的 α-突触核蛋白（α-synuclein(gT72)）的合成方案。然而，这种利用两个重组片段和一个合成肽的通用方法适用于其他位点和类型的修饰，并且应该可以转移到各种其他蛋白质靶标，包括聚集倾向蛋白，如 tau 和 TDP-43。
