Table_1_Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification.xlsx

Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.

重组蛋白表达已成为基础与应用研究中不可或缺的核心工具。该领域积累的研究知识已支持数千个蛋白质靶点以可溶性、高纯度且均一的状态实现表达，这是生化分析与结构解析的必要前提。近数十年来该领域取得了显著进展：通过优化宿主、菌株、融合伴侣、启动子强度等多种参数，此前难以表达的挑战性蛋白已可实现可溶性表达。有鉴于此，我们团队此前开发了一套载体套件，可通过简便高效的克隆策略，实现不同启动子与可溶性增强蛋白的筛选评估。尽管如此，仍有大量蛋白质靶点的高效表达难以实现，例如需要引入复杂的翻译后修饰（post-translational modifications），或需在特定细胞区室中完成表达。为突破单一亚细胞定位与单一宿主类型带来的研究局限，本文中我们对此前开发的载体套件进行了拓展，新增了重组蛋白在不同细胞区室与宿主细胞中的表达评估功能。此外，该系列载体还可用于筛选替代性纯化策略，以提升目标蛋白的得率。