Department of Chemistry, Princeton University, Frick Laboratory, Princeton, New Jersey 08544, United States.
J Am Chem Soc. 2013 Jan 9;135(1):286-92. doi: 10.1021/ja309126m. Epub 2012 Dec 24.
Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.
化学修饰的蛋白质是研究生物过程分子细节的宝贵工具，它们也具有作为新型治疗剂的巨大潜力。已经开发了几种用于蛋白质定点修饰的方法，其中最广泛使用的是表达蛋白连接（EPL），其中重组α-硫酯与含有 N 端半胱氨酸的肽连接。尽管 EPL 得到了广泛应用，但所需重组蛋白α-硫酯的产生和分离仍然具有挑战性。我们在这里描述了一种使用工程化的天然分裂 DnaE 内含子制备和纯化重组蛋白α-硫酯的新方法。这一家族的自动加工酶与目前用于生成蛋白α-硫酯的内含子密切相关，但它们具有更快的动力学，并且分裂成两个需要关联才能变得活跃的非活性多肽。利用两个分裂内含子片段之间的强亲和力，我们设计了一种从细胞裂解物中纯化和生成蛋白α-硫酯的简化程序，并将该策略应用于各种蛋白质的半合成，包括乙酰化组蛋白和特异性修饰的单克隆抗体。
