Lawrie Justin, Niu Wei, Guo Jiantao
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
Department of Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, United States.
Methods Enzymol. 2019;622:67-89. doi: 10.1016/bs.mie.2019.02.004. Epub 2019 Mar 4.
Protein tyrosine O-sulfation is considered as one of the most common types of posttranslational modification of tyrosine in nature. The introduction of a negatively charged sulfate group plays crucial roles in extracellular biomolecular interactions that dictate various cellular processes, including cell adhesion, leukocyte trafficking, hormone activities, and immune responses. Despite substantial advances in our knowledge about protein tyrosine O-sulfation in recent years, our understanding of its biological significance is still in its infancy. This is largely hindered by a chronic lack of suitable biochemical tools. We seek to meet this challenge by engineering a small protein scaffold that can recognize sulfated tyrosine (sulfotyrosine) residues with high affinity. In this chapter, we describe the directed evolution of a Src Homology 2 (SH2) domain to recognize sulfotyrosine. In the first part, the design strategy for the phage display of SH2 variants is discussed. In the second part, the techniques required for phage propagation and selection are described. The evolved SH2 variants are characterized and validated in vitro through fluorescence polarization assays. Finally, the evolved SH2 domain mutants are applied to the visualization of sulfated proteins on the cell surface.
蛋白质酪氨酸O-硫酸化被认为是自然界中酪氨酸最常见的翻译后修饰类型之一。带负电荷的硫酸基团的引入在决定各种细胞过程的细胞外生物分子相互作用中起着关键作用，这些过程包括细胞粘附、白细胞运输、激素活性和免疫反应。尽管近年来我们对蛋白质酪氨酸O-硫酸化的认识有了很大进展，但我们对其生物学意义的理解仍处于起步阶段。这在很大程度上受到长期缺乏合适生化工具的阻碍。我们试图通过构建一种能够高亲和力识别硫酸化酪氨酸（硫酪氨酸）残基的小蛋白质支架来应对这一挑战。在本章中，我们描述了一种Src同源2（SH2）结构域识别硫酪氨酸的定向进化。第一部分讨论了SH2变体噬菌体展示的设计策略。第二部分描述了噬菌体繁殖和筛选所需的技术。通过荧光偏振分析在体外对进化后的SH2变体进行表征和验证。最后，将进化后的SH2结构域突变体应用于细胞表面硫酸化蛋白的可视化。
