Weber Alain R, Schuermann David, Schär Primo
Department of Biomedicine, University of Basel, Basel, Switzerland.
PLoS One. 2014 Jul 9;9(7):e102157. doi: 10.1371/journal.pone.0102157. eCollection 2014.
Posttranslational modification by small ubiquitin-like modifiers (SUMO) is being associated with a growing number of regulatory functions in diverse cellular processes. The biochemical investigation into the underlying molecular mechanisms, however, has been lagging behind due to the difficulty to generate sufficient amounts of recombinant SUMOylated proteins. Here, we present two newly designed two-component vector systems for the expression and purification of SUMO-modified target proteins in Escherichia coli. One system consists of a vector for SUMO conjugation, expressing human SUMO-activating (SAE1/SAE2) and conjugating (Ubc9) enzymes together with His6-tagged SUMO1, 2 or 3, that can be combined with commonly used expression constructs for any gene of interest. To facilitate SUMOylation of targets normally requiring a SUMO-E3 ligase for efficient modification, a second system is designed to express the target protein as a fusion with the human SUMO-conjugating enzyme Ubc9, thus compensating the absence of a potential SUMO ligase. We demonstrate the proficiency of these systems by SUMOylation of two DNA repair proteins, the thymine DNA glycosylase (TDG) and XRCC1, and describe purification schemes for SUMOylated proteins in native and active form. This SUMO toolbox facilitates "in-cell" and "in-extract" production and purification of recombinant SUMO-modified target proteins for functional and structural analysis.
小泛素样修饰物（SUMO）介导的翻译后修饰与多种细胞过程中越来越多的调节功能相关。然而，由于难以产生足够量的重组SUMO化蛋白，对其潜在分子机制的生化研究一直滞后。在此，我们展示了两种新设计的双组分载体系统，用于在大肠杆菌中表达和纯化SUMO修饰的靶蛋白。一种系统由一个用于SUMO缀合的载体组成，该载体表达人SUMO激活酶（SAE1/SAE2）和缀合酶（Ubc9）以及His6标记的SUMO1、SUMO2或SUMO3，它可以与用于任何感兴趣基因的常用表达构建体相结合。为了促进通常需要SUMO-E3连接酶进行有效修饰的靶标的SUMO化，设计了第二种系统，将靶蛋白与人SUMO缀合酶Ubc9融合表达，从而弥补潜在SUMO连接酶的缺失。我们通过对两种DNA修复蛋白——胸腺嘧啶DNA糖基化酶（TDG）和XRCC1进行SUMO化，证明了这些系统的有效性，并描述了天然和活性形式的SUMO化蛋白的纯化方案。这个SUMO工具箱有助于“细胞内”和“提取物中”重组SUMO修饰靶蛋白的生产和纯化，用于功能和结构分析。
