Schulich Faculty of Chemistry, Technion-Israel Institute of Technology Haifa 3200008, Israel.
Bioconjug Chem. 2020 Mar 18;31(3):889-894. doi: 10.1021/acs.bioconjchem.0c00026. Epub 2020 Feb 24.
Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a β-grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.
干扰素刺激基因 15（ISG15）是泛素样修饰物（ULM）家族的一员，它采用类似于泛素（Ub）的β-抓握折叠结构域（s），只有微小的序列同源性。ISG15 由两个 Ub 样结构域组成，有助于免疫系统中和多种病原体的感染，并在抵御包括甲型流感在内的许多病毒方面发挥重要作用。最近，发现 Ub 是 ISG15 的底物，它可以在 Lys29 和 Lys48 上被 ISG 化，而前者更为主要。这种混合的 ISG15-Ub 链的发现提出了关于这种缀合的性质和效果的各种基本问题。为了进一步研究混合 ISG15-Ub 链的作用，需要在可行的数量下获得这些链的纯均相材料。通过应用先进的蛋白质合成化学策略，我们报告了 231 个残基的 ISG15-Lys29-Ub 混合链的全化学合成。在合成过程中，我们遇到了不溶性肽片段，因此我们开发了一种新的基于可逆 Acm 的可溶性标记物来有效地解决这个问题。这种新的 Acm 标记物与已知的基于 Arg 的 Acm 可溶性标记物进行了比较，并且在合成天然 ISG15-Ub 混合链时，在掺入和效率方面被发现更可靠。
