School of Biological Science, Nanyang Technological University, Singapore, Singapore.
Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Zhejiang, China.
Methods Mol Biol. 2022;2530:177-193. doi: 10.1007/978-1-0716-2489-0_13.
Peptidyl Asx-specific ligases (PALs) effect peptide ligation by catalyzing transpeptidation reactions at Asn/Asp-peptide bonds. Owing to their high efficiency and mild aqueous reaction conditions, these ligases have emerged as powerful biotechnological tools for protein manipulation in recent years. PALs are enzymes of the asparaginyl endopeptidase (AEP) superfamily but have predominant transpeptidase activity as opposed to typical AEPs which are predominantly hydrolases. Butelase-1 and VyPAL2, two PALs discovered by our teams, have been used successfully in a wide range of applications, including macrocyclization of synthetic peptides and recombinant proteins, protein N- or C-terminal modification, and cell-surface labeling. As shown in numerous reports, PAL-mediated ligation is highly efficient at Asn junctions. Although considerably less efficient, Asp-specific ligation has also been shown to be practically useful under suitable conditions. Herein, we describe the methods of using VyPAL2 for protein macrocyclization and labeling at an Asp residue as well as for protein dual labeling through orthogonal Asp- and Asn-directed ligations. We also describe a method for cell-surface protein modification using butelase-1, demonstrating its advantageous features over previous methods.
天冬酰氨专一性连接酶（PALs）通过催化天冬酰氨/天冬氨酸肽键的转肽反应来实现肽键的连接。由于其高效性和温和的水相反应条件，近年来这些连接酶已成为蛋白质操作的强大生物技术工具。PALs 是天冬酰胺内肽酶（AEP）超家族的酶，但与典型的 AEP 相比，它们具有明显的转肽酶活性，而典型的 AEP 主要是水解酶。Butelase-1 和 VyPAL2 是我们团队发现的两种 PAL，已成功应用于广泛的应用中，包括合成肽和重组蛋白的大环化、蛋白质 N 或 C 末端修饰以及细胞表面标记。正如许多报道所表明的那样，PAL 介导的连接在天冬酰氨连接处具有很高的效率。尽管效率要低得多，但在合适的条件下，天冬氨酸特异性连接也被证明是实用的。本文描述了使用 VyPAL2 进行蛋白质大环化和 Asp 残基标记以及通过正交的 Asp 和 Asn 导向连接进行蛋白质双重标记的方法。我们还描述了一种使用 butelase-1 进行细胞表面蛋白修饰的方法，展示了其相对于先前方法的优势特征。
