School of Biological Sciences, University of Reading, Reading RG6 6UR, UK.
Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.
Int J Mol Sci. 2021 Sep 10;22(18):9805. doi: 10.3390/ijms22189805.
The bacterial pathogen, , has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised cloned in three different expression plasmids was examined in a library of host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised . Transcriptional- reporter fusions defined the P promoter and Caf1R binding site responsible for transcription of the operon. Use of the identified Caf1R binding DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of .
细菌病原体 已经引发了三次历史性的大流行，并继续在全球范围内引发小规模疫情爆发。在感染过程中， 会组装出一层由 Caf1 亚基细纤维组成的类似胶囊的保护性外壳。由于其在鼠疫诊断和抗鼠疫疫苗开发中的临床价值，这种 F1 荚膜抗原引起了广泛关注。F1 的表达受到 AraC/XylS 家族转录激活因子 Caf1R 的严格调控，这种蛋白因易于聚集而臭名昭著。在这里，我们对可溶性 Caf1R 的重组表达进行了优化。在宿主菌株文库中，分别从天然和合成的、克隆在三种不同表达质粒中的 进行了表达。体内证明了 His 标记的 Caf1R 的功能，但过表达时出现不溶性问题。从密码子优化的 中大量产生了可溶性 MBP-Caf1R。转录报告基因融合定义了 P 启动子和 Caf1R 结合位点，负责 操纵子的转录。在电泳迁移率变动分析 (EMSA) 中使用鉴定出的 Caf1R 结合 DNA 序列证实了重组 MBP-Caf1R 中 Caf1R DNA 结合结构域的正确折叠和功能。功能性重组 Caf1R 的可用性将是阐明 F1 表达控制和 Caf1R 调节的 病理生理学的有价值的工具。
