Afful Derrick, Cai Liming, Momany Cory
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA, 30602, USA.
Department of Computer Sciences, University of Georgia, Athens, GA, 30602, USA.
Protein Expr Purif. 2019 Nov;163:105448. doi: 10.1016/j.pep.2019.105448. Epub 2019 Jul 4.
The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is the engine of gene expression. Despite global conservation in their structures and function, RNAPs from different bacteria can have unique features in promoter and transcription factor recognition. Therefore, availability of purified RNAP from different bacteria is key to understanding these species-specific aspects and will be valuable for antibiotic drug discovery. Pseudomonas aeruginosa is one of the leading causes of hospital and community acquired infections worldwide - making the organism an important public health pathogen. We developed a method for producing high quantities of highly pure and active recombinant P. aeruginosa str. PAO1 RNAP core and holoenzyme complexes that employed two-vector systems for expressing the core enzyme (α, β, β', and ω subunits) and for expressing the holoenzyme complex (core + σ). Unlike other RNAP expression approaches, we used a low temperature autoinduction system in E. coli with T7 promoters that produced high cell yields and stable protein expression. The purification strategy comprised of four chromatographic separation steps (metal chelate, heparin, and ion-exchange) with yields of up to 11 mg per 500 mL culture. Purified holoenzyme and reconstituted holoenzyme from core and σ were highly active at transcribing both small and large-sized DNA templates, with a determined elongation rate of ~18 nt/s for the holoenzyme. The successful purification of the P. aeruginosa RNAP provides a gateway for studies focusing on in vitro transcriptional regulation in this pathogen.
细菌RNA聚合酶（RNAP）是一种大型复杂的分子机器，是基因表达的引擎。尽管不同细菌的RNAP在结构和功能上具有全球保守性，但来自不同细菌的RNAP在启动子和转录因子识别方面可能具有独特特征。因此，获得来自不同细菌的纯化RNAP是理解这些物种特异性方面的关键，并且对于抗生素药物发现将具有重要价值。铜绿假单胞菌是全球医院获得性感染和社区获得性感染的主要原因之一，使该生物体成为一种重要的公共卫生病原体。我们开发了一种方法，用于大量生产高纯度和活性的重组铜绿假单胞菌菌株PAO1 RNAP核心酶和全酶复合物，该方法采用双载体系统来表达核心酶（α、β、β'和ω亚基）以及表达全酶复合物（核心酶+σ因子）。与其他RNAP表达方法不同，我们在大肠杆菌中使用了带有T7启动子的低温自诱导系统，该系统产生了高细胞产量和稳定的蛋白质表达。纯化策略包括四个色谱分离步骤（金属螯合、肝素和离子交换），每500 mL培养物的产量高达11 mg。纯化的全酶以及由核心酶和σ因子重构的全酶在转录小型和大型DNA模板时都具有高活性，全酶的测定延伸速率约为18 nt/s。成功纯化铜绿假单胞菌RNAP为专注于该病原体体外转录调控的研究提供了途径。
