Taschner Michael, Dickinson Joe Bradley, Roisné-Hamelin Florian, Gruber Stephan
Department of Fundamental Microbiology (DMF), Faculty of Biology and Medicine (FBM), University of Lausanne (UNIL), Lausanne, Switzerland
Department of Fundamental Microbiology (DMF), Faculty of Biology and Medicine (FBM), University of Lausanne (UNIL), Lausanne, Switzerland.
Life Sci Alliance. 2024 Dec 2;8(1). doi: 10.26508/lsa.202402899. Print 2025 Jan.
Multisubunit protein complexes are central to many cellular processes, and studying their activities and structures in vitro requires reconstitution via recombinant expression and purification. Obtaining targets at sufficient purity and scale typically involves screening several protein variants and expression hosts. Existing cloning strategies enable co-expression but are often time-consuming, labor-intensive, and host-specific, or involve error-prone steps. We present a novel vector set and assembly strategy to overcome these limitations, enabling expression construct generation for multisubunit complexes in a single step. This modular system can be extended to additional hosts or include new tags. We demonstrate its utility by constructing expression vectors for structural maintenance of chromosomes complexes in various hosts, streamlining workflows, and improving productivity.
多亚基蛋白质复合物是许多细胞过程的核心，在体外研究它们的活性和结构需要通过重组表达和纯化来进行重构。以足够的纯度和规模获得目标蛋白通常需要筛选几种蛋白质变体和表达宿主。现有的克隆策略能够实现共表达，但往往耗时、费力且具有宿主特异性，或者涉及容易出错的步骤。我们提出了一种新颖的载体集和组装策略来克服这些限制，能够一步生成多亚基复合物的表达构建体。这个模块化系统可以扩展到其他宿主或包含新的标签。我们通过在各种宿主中构建用于染色体复合物结构维持的表达载体、简化工作流程并提高生产力来证明其效用。
