Biochemistry Center (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
Int J Mol Sci. 2022 Mar 16;23(6):3198. doi: 10.3390/ijms23063198.
is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50-52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications.
是一种有吸引力的真核模式生物，由于其异常高的温度耐受性（最佳生长温度为 50-52°C），具有热稳定的蛋白质组，可用于生化、结构和生物技术应用。为了研究耐热蛋白的结构和功能及其在复合物中的组织，标记靶基因的定点基因操作是一种理想的方法，但尚未在这种嗜热菌中建立。在这里，我们描述了一种同源重组系统的开发，用于在 中对感兴趣的染色体重组基因进行表位标记，目的是利用衍生的耐热融合蛋白进行串联亲和纯化。通过工程合适的菌株，消除非同源末端连接途径的因素，从而提高特定基因座同源整合的效率，促进了这种遗传方法的发展。通过这种策略，我们可以证明通过同源重组进行基因标记可以提高纯化诱饵蛋白和共沉淀因子的产量，为基础研究和工业应用中的相关研究铺平了道路。
