György Babnigg, Robert Jedrzejczak, Boguslaw Nocek, Adam Stein, William Eschenfeldt, Lucy Stols, Norman Marshall, Alicia Weger, Ruiying Wu, Mark Donnelly, Andrzej Joachimiak
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引用次数: 0
Abstract
Multiprotein complexes play essential roles in all cells and X-ray crystallography can provide unparalleled insight into their structure and function. Many of these complexes are believed to be sufficiently stable for structural biology studies, but the production of protein-protein complexes using recombinant technologies is still labor-intensive. We have explored several strategies for the identification and cloning of heterodimers and heterotrimers that are compatible with the high-throughput (HTP) structural biology pipeline developed for single proteins. Two approaches are presented and compared which resulted in co-expression of paired genes from a single expression vector. Native operons encoding predicted interacting proteins were selected from a repertoire of genomes, and cloned directly to expression vector. In an alternative approach, Helicobacter pylori proteins predicted to interact strongly were cloned, each associated with translational control elements, then linked into an artificial operon. Proteins were then expressed and purified by standard HTP protocols, resulting to date in the structure determination of two H. pylori complexes.
多蛋白复合物在所有细胞中都发挥着至关重要的作用,而 X 射线晶体学可以提供对其结构和功能的无与伦比的洞察力。据信,这些复合物中有许多在结构生物学研究中足够稳定,但利用重组技术生产蛋白质-蛋白质复合物仍是一项劳动密集型工作。我们探索了几种识别和克隆异源二聚体和异源三聚体的策略,这些策略与为单个蛋白质开发的高通量(HTP)结构生物学流水线兼容。本文介绍了两种方法并进行了比较,这两种方法通过单一表达载体实现了成对基因的共同表达。从基因组库中选择了编码预测的相互作用蛋白的本地操作子,并直接克隆到表达载体上。另一种方法是克隆幽门螺旋杆菌蛋白质,预测它们会发生强烈的相互作用,每种蛋白质都与翻译控制元件相关联,然后连接到人工操作子中。然后用标准的 HTP 方案表达和纯化蛋白质,迄今已确定了两种幽门螺杆菌复合物的结构。