用于生物物理研究的大肠杆菌III型毒素-抗毒素核糖核蛋白复合物及其组分的大规模纯化。

Parthasarathy Manikandan, Kavyashree Nadig, Mahavir Singh
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引用次数: 1

摘要

毒素-抗毒素(TA)系统是广泛存在的细菌免疫系统,可以保护细菌免受各种环境压力的侵害。根据抗毒素的性质和作用机制,将TA系统分为八类(I-VIII)。III型TA系统由非编码RNA抗毒素和蛋白质毒素组成,形成核糖核蛋白(RNP) TA复合物,在细菌噬菌体防御中起关键作用。III型TA系统存在于人类肠道微生物群和几种致病菌中,因此可以用于新型抗菌策略。由于毒素对大肠杆菌的固有毒性,从大肠杆菌表达系统中过表达和纯化游离毒素具有挑战性。因此,蛋白质毒素通常与抗毒素RNA作为RNP复合物从大肠杆菌中共表达和共纯化,用于结构和生物物理研究。在这里,我们优化了大肠杆菌毒素III型TA复合物的共表达和纯化方法,从而纯化了TA RNP复合物,并且通常可以获得生物物理和结构研究所需的游离抗毒素RNA和游离活性毒素。该方案也可以适用于纯化同位素标记(例如,均匀的15N-或13c标记)的游离毒素蛋白,游离抗毒素rna和TA RNPs,这些可以使用多维核磁共振(NMR)波谱方法进行研究。主要特点大肠杆菌III型毒素-抗毒素复合物大规模纯化的详细方案。优化后的方案可获得数毫克的TA RNP复合物、游离毒素和游离抗毒素RNA。在获得所需的DNA克隆后,使用市售质粒载体和化学品在5天内完成该方案。纯化的TA复合物、毒素蛋白和抗毒素RNA用于生物物理实验,如NMR、ITC和x射线晶体学。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from <i>Escherichia coli</i> for Biophysical Studies.

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from <i>Escherichia coli</i> for Biophysical Studies.

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from <i>Escherichia coli</i> for Biophysical Studies.

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from Escherichia coli for Biophysical Studies.

Toxin-antitoxin (TA) systems are widespread bacterial immune systems that confer protection against various environmental stresses. TA systems have been classified into eight types (I-VIII) based on the nature and mechanism of action of the antitoxin. Type III TA systems consist of a noncoding RNA antitoxin and a protein toxin, forming a ribonucleoprotein (RNP) TA complex that plays crucial roles in phage defence in bacteria. Type III TA systems are present in the human gut microbiome and several pathogenic bacteria and, therefore, could be exploited for a novel antibacterial strategy. Due to the inherent toxicity of the toxin for E. coli, it is challenging to overexpress and purify free toxins from E. coli expression systems. Therefore, protein toxin is typically co-expressed and co-purified with antitoxin RNA as an RNP complex from E. coli for structural and biophysical studies. Here, we have optimized the co-expression and purification method for ToxIN type III TA complexes from E. coli that results in the purification of TA RNP complex and, often, free antitoxin RNA and free active toxin in quantities required for the biophysical and structural studies. This protocol can also be adapted to purify isotopically labelled (e.g., uniformly 15N- or 13C-labelled) free toxin proteins, free antitoxin RNAs, and TA RNPs, which can be studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy methods. Key features Detailed protocol for the large-scale purification of ToxIN type III toxin-antitoxin complexes from E. coli. The optimized protocol results in obtaining milligrams of TA RNP complex, free toxin, and free antitoxin RNA. Commercially available plasmid vectors and chemicals are used to complete the protocol in five days after obtaining the required DNA clones. The purified TA complex, toxin protein, and antitoxin RNA are used for biophysical experiments such as NMR, ITC, and X-ray crystallography.

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