School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Protein Expr Purif. 2023 Dec;212:106351. doi: 10.1016/j.pep.2023.106351. Epub 2023 Aug 20.
Vesicle trafficking is a fundamental cellular process that ensures proper material exchange between organelles in eukaryotic cells, and multisubunit tethering complexes (MTCs) are essential in this process. The heterohexameric homotypic fusion and protein sorting (HOPS) complex, which functions in the endolysosomal pathway, is a member of MTCs. Despite its critical role, the complex composition and low-expression level of HOPS have made its expression and purification extremely challenging. In this study, we present a highly efficient strategy for overexpressing and purifying HOPS from Saccharomyces cerevisiae. We achieved HOPS overexpression by integrating a strong promoter TEF1 before each subunit using the gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) system. The HOPS complex was subsequently purified using Staphylococcus aureus protein A (ProtA) affinity purification and size-exclusion chromatography, resulting in high purity and homogeneity. We obtained two-fold more HOPS using this method than that obtained using the commonly used GAL1 promoter-controlled HOPS overexpression. Negative staining electron microscopy analysis confirmed the correct assembly of HOPS. Notably, we also successfully purified two other MTCs, class C core vacuole/endosome tethering (CORVET) and Golgi-associated retrograde protein (GARP) using this approach. Our findings facilitate further in vitro biochemical characterization and functional studies of MTCs and provide a useful guide for the preparation of other heterogenic multisubunit complexes.
囊泡运输是真核细胞中确保细胞器之间物质交换的基本细胞过程，多亚基 tethering 复合物（MTCs）在这个过程中是必不可少的。在这个过程中，发挥作用的异六聚体同源融合和蛋白分拣（HOPS）复合物是 MTCs 的成员之一。尽管其具有重要作用，但由于 HOPS 的复杂组成和低表达水平，其表达和纯化极具挑战性。在本研究中，我们提出了一种从酿酒酵母中高效表达和纯化 HOPS 的策略。我们通过使用 gRNA-tRNA 阵列的 CRISPR-Cas9（GTR-CRISPR）系统，在每个亚基前整合一个强启动子 TEF1 来实现 HOPS 的过表达。HOPS 复合物随后使用金黄色葡萄球菌蛋白 A（ProtA）亲和纯化和分子筛层析进行纯化，得到高纯度和均一性。与使用常用的 GAL1 启动子控制的 HOPS 过表达相比，我们使用这种方法获得了两倍多的 HOPS。负染色电子显微镜分析证实了 HOPS 的正确组装。值得注意的是，我们还使用这种方法成功地纯化了另外两种 MTCs，即 C 类核心液泡/内体 tethering（CORVET）和高尔基体相关逆行蛋白（GARP）。我们的研究结果为 MTCs 的进一步体外生化特性和功能研究提供了便利，并为其他异质多亚基复合物的制备提供了有用的指导。
