The linear ubiquitin chain assembly complex tethering motif (LUBAC-LTM) domain is composed of two different accessory LUBAC components (HOIL-1L and SHARPIN) but folds as a single globular domain. Targeted disruption of the intricate LTM-LTM interaction destabilizes LUBAC in lymphoma cells, thereby attenuating LUBAC stability, which highlights that targeting the interaction between the two LTM motifs is a promising strategy for the development of new agents against cancers that depend on LUBAC activity for their survival. To further screen for small-molecule inhibitors that can selectively disrupt the LTM-LTM interaction, it is necessary to obtain high-purity samples of the LTM domain. Ideally, such a sample would not contain any components other than the LTM itself, so that false positives (molecules binding to other parts of LUBAC) could be eliminated from the screening process. Here we report a simple strategy that enabled successful bacterial production of the isolated LUBAC LTM domain in high yield and at high purity. The strategy combines (1) structural analysis highlighting the possibility of tandem expression in the SHARPIN^L™ to HOIL-1L^L™ direction; (2) bacterial expression downstream of EGFP to efficiently monitor expression and solubility; (3) gentle low-temperature folding using autoinduction. Formation of stably folded LTM was verified by size-exclusion chromatography and heteronuclear NMR spectroscopy. From 200-ml cultures sufficient quantities (~7 mg) of high-purity protein for structural studies could be obtained. The presented strategy will be beneficial for LUBAC LTM-based drug-screening efforts and likely serve as a useful primer for similar cases, i.e., whenever a smaller folded fragment is to be isolated from a larger protein complex for site-specific downstream applications.

线性泛素链组装复合物连接基序( LUBAC - LTM) 域由两个不同的附属 LUBAC 组件（HOIL-1L 和 SHARPIN）组成，但折叠为单个球状域。靶向破坏复杂的 LTM-LTM 相互作用会破坏淋巴瘤细胞中 LUBAC 的稳定性，从而减弱 LUBAC 稳定性，这突出表明，靶向两个 LTM 基序之间的相互作用是开发针对依赖 LUBAC 活性的癌症的新药物的有希望的策略。生存。为了进一步筛选可以选择性破坏 LTM-LTM 相互作用的小分子抑制剂，有必要获得 LTM 结构域的高纯度样品。理想情况下，这样的样本不会包含除 LTM 本身以外的任何成分，因此可以从筛选过程中消除假阳性（与 LUBAC 其他部分结合的分子）。在这里，我们报告了一种简单的策略，该策略能够以高产率和高纯度成功地细菌生产分离的 LUBAC LTM 结构域。该策略结合了 (1) 结构分析，突出了^{SHARPIN L} ™ 到 HOIL-1L ^L ™ 方向的串联表达；(2) EGFP 下游的细菌表达，以有效监测表达和溶解度；(3) 使用自动感应的温和低温折叠。通过尺寸排阻色谱和异核 NMR 光谱验证了稳定折叠 LTM 的形成。从 200-ml 培养物中可以获得足量（~7mg）的高纯度蛋白质用于结构研究。所提出的策略将有利于基于 LUBAC LTM 的药物筛选工作，并可能作为类似情况的有用引物，即，每当要从较大的蛋白质复合物中分离较小的折叠片段以用于特定位点的下游应用时。