Abstract：
Employing biophysical and structural methods is a powerful way to elucidate mechanisms of molecular recognition in bacterial pathogenesis. Such studies invariably depend on the production of pure, folded and stable proteins. Many proteins that can be expressed recombinantly ultimately fail to meet one or more of these criteria. The cag proteins from Helicobacter pylori form a secretion system that delivers the oncoprotein, CagA, into human gastric epithelial cells through an interaction between CagL and host cell integrins, where it can cause gastric adenocarcinoma. Expression of full length CagA and CagL is problematic as CagA undergoes rapid degradation during purification and CagL is thermally unstable. Here, we describe a method for the purification of CagA that results in the production of the full length protein through coexpression with its endogenous chaperone, CagF, and its subsequent separation from its chaperone. Furthermore, we detail the production of CagL and the use of differential scanning fluorimetry to identify how CagL is thermally stabilized by reduced pH, which led to a new crystal form of CagL and novel insight to pathogenic mechanisms. The methods described here for the production of stable cag proteins can be applied to a wide range of proteins involved in bacterial pathogenesis.
摘要：
采用生物物理和结构方法是阐明细菌发病机理中分子识别机制的有力方法。这样的研究总是依赖于生产纯净的，折叠和稳定的蛋白质。可以重组表达的许多蛋白质最终不能满足这些标准中的一个或多个。来自幽门螺杆菌的cag蛋白形成一个分泌系统，传递癌蛋白，卡加,通过CagL和宿主细胞整合素之间的相互作用进入人胃上皮细胞，会导致胃腺癌.全长CagA和CagL的表达是有问题的,因为CagA在纯化期间经历快速降解并且CagL是热不稳定的。这里,我们描述了一种纯化CagA的方法，该方法通过与其内源性伴侣共表达来产生全长蛋白，CagF,以及随后与伴侣的分离。此外，我们详细介绍了CagL的生产和差示扫描荧光分析法的使用，以确定如何通过降低pH来使CagL热稳定。这导致了CagL的新晶体形式和对致病机制的新见解。这里描述的用于生产稳定cag蛋白的方法可以应用于涉及细菌发病机理的多种蛋白。
