Shanghai Fifth People's Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.
Shanghai Fifth People's Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.
Protein Expr Purif. 2023 Oct;210:106318. doi: 10.1016/j.pep.2023.106318. Epub 2023 Jun 5.
Kelch-like protein 6 (KLHL6) plays a critical role in preventing the development and survival of diffuse large B-cell lymphoma (DLBCL) through its involvement in the ubiquitin proteasome system. Specifically, KLHL6 binds to cullin3 (Cul3) and the substrate, facilitating the assembly of the E3 ligase responsible for substrate ubiquitination. It is imperative to investigate the precise function of KLHL6 by conducting a structural analysis of its interaction with Cul3. Here, we present the expression, purification, and characterization of the full-length KLHL6. Our findings demonstrate that the addition of a Sumo-tag significantly enhances the production of KLHL6, while also improving its stability and solubility. Moreover, through gel filtration and negative staining electron microscopy (EM), we observed that KLHL6 adopts a homomultimeric form in solution. Additionally, we found that the presence of Cul3 enhances the stability and homogeneity of KLHL6 by forming a complex. Consequently, the successful expression and purification of full-length KLHL6 serve as a foundation for further investigations into the structure and function of the KLHL6/Cullin3/Rbx1 substrate complex, as well as provide a potential strategy for studying other proteins within the KLHL family that possess similar characteristics.
Kelch 样蛋白 6（KLHL6）通过参与泛素蛋白酶体系统在预防弥漫性大 B 细胞淋巴瘤（DLBCL）的发生和存活方面起着至关重要的作用。具体来说，KLHL6 与 cullin3（Cul3）和底物结合，促进负责底物泛素化的 E3 连接酶的组装。通过对 KLHL6 与 Cul3 的相互作用进行结构分析，研究 KLHL6 的精确功能至关重要。在这里，我们展示了全长 KLHL6 的表达、纯化和表征。我们的研究结果表明，添加 Sumo 标签可显著提高 KLHL6 的产量，同时还提高其稳定性和溶解度。此外，通过凝胶过滤和负染电子显微镜（EM）观察到 KLHL6 在溶液中采用同型多聚体形式。此外，我们发现 Cul3 的存在通过形成复合物增强了 KLHL6 的稳定性和均一性。因此，全长 KLHL6 的成功表达和纯化为进一步研究 KLHL6/Cullin3/Rbx1 底物复合物的结构和功能奠定了基础，并为研究具有相似特征的 KLHL 家族中的其他蛋白质提供了一种潜在策略。
