CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark.
Sci Rep. 2017 Jan 18;7:40756. doi: 10.1038/srep40756.
Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.
泛素和泛素样蛋白（UbLs）的翻译后修饰对于维持蛋白质的内稳态至关重要。UbL 缀合物的有效分离受到多种因素的阻碍，包括试剂的成本和特异性、蛋白酶对 UbLs 的去除、UbL 缀合物与相互作用物的区分以及修饰底物的低丰度。在这里，我们描述了 bioUbLs，这是一组基于多顺反子表达和大肠杆菌生物素蛋白连接酶 BirA 的体内生物素化的工具，用于研究果蝇和哺乳动物中的修饰。虽然 bioUbLs 允许快速验证外源或内源蛋白的 UbL 缀合，但单个载体方法可以促进大多数感兴趣的蛋白质的生物素化。变性条件下的纯化使去缀合酶失活，严格的洗涤去除 UbL 相互作用物和非特异性背景。我们在果蝇细胞和转基因果蝇中证明了该方法的实用性，在这两种情况下都鉴定出了大量推定的 SUMO 化蛋白。对于哺乳动物细胞，我们展示了许多不同 UbLs 的缀合和定位，并鉴定了 UFM1 的新潜在底物。使用方便且能够修改现有载体，使得 bioUbL 系统成为研究这种重要蛋白质调控方式的现有策略的有力补充。
