Popp Maximilian Wei-Lin
Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave, Rochester, NY, 14642, USA,
Methods Mol Biol. 2015;1266:185-98. doi: 10.1007/978-1-4939-2272-7_13.
Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant.
位点特异性标记蛋白质生物共轭物的构建对于分子生物学家和细胞生物学家而言是一项重要工具。化学标记方法虽然在可连接的基团类型方面具有通用性，但缺乏特异性，常常靶向蛋白质内的多个位置。在此，我们描述了使用细菌转肽酶分选酶A在蛋白质C末端进行化学酶法标记的方案。我们详细介绍了一种用于纯化金黄色葡萄球菌酶（SrtA 5(o)）改良五突变体变体的方案，该变体相对于野生型酶展现出大幅改善的动力学特性。重要的是，还描述了一种使用可适用于任何没有现有合成化学基础设施的细胞/分子生物学实验室的技术构建与分选酶标记兼容的肽探针的方案。最后，我们给出了一个如何使用改良的SrtA 5(o)变体优化标记反应的示例。
