Picking W L, Mertz J A, Marquart M E, Picking W D
Department of Biology, Saint Louis University, Missouri 63103-2010, USA.
Protein Expr Purif. 1996 Dec;8(4):401-8. doi: 10.1006/prep.1996.0117.
Shigella flexneri and related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems from S. flexneri invasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid of S. flexneri that have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of the ipa operon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailed in vitro analysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins from Escherichia coli for use in dissecting of the protein biochemistry of S. flexneri pathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response to S. flexneri invasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasions, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.
福氏志贺菌及相关肠道致病菌是导致细菌性痢疾的重要病原体，在世界不发达地区，细菌性痢疾对儿童来说可能是一种危及生命的疾病。志贺菌病的发病源于福氏志贺菌对结肠上皮细胞的侵袭，导致局部细胞死亡和炎症。侵袭质粒抗原（Ipa）B、C和D是福氏志贺菌大毒力质粒编码的三种分泌蛋白，被认为是这种细胞侵袭过程的关键效应因子。这些蛋白作为ipa操纵子的一部分表达，是宿主对志贺菌病免疫反应的主要靶点之一。由于Ipa侵袭素没有以详细体外分析所需的量进行纯化，其生化特性的表征一直很复杂。在此，我们描述了首次从大肠杆菌中克隆、表达并大量纯化IpaB和IpaC融合蛋白，用于剖析福氏志贺菌致病机制中的蛋白质生物化学。我们采用了多种方法以可溶形式大量制备这些蛋白，包括使用不同的宿主细胞系、改变细菌生长条件以及使用替代质粒表达载体。既然这些Ipa蛋白已以高纯度形式获得，就有可能启动对其重要生物学和免疫学特性以及它们募集到高分子量蛋白复合物中的研究。与IpaD（作为先前一项研究的一部分进行了纯化）一起，这些纯化的蛋白将有助于：（a）探索宿主对福氏志贺菌侵袭的免疫反应特性；（b）阐明导致病原体内化的具体生化特性；（c）分析特定Ipa蛋白复合物在宿主细胞侵袭中的重要性；（d）监测甚至增强人类试验中口服活疫苗的效力。
