ePrints@IIScePrints@IISc Home | About | Browse | Latest Additions | Advanced Search | Contact | Help

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

Manikandan, P and Nadig, K and Singh, M (2023) Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from Escherichia coli for Biophysical Studies. In: Bio-protocol, 13 (13).

[img]
Preview
PDF
bio_pro_13-13_2023.pdf - Published Version

Download (983kB) | Preview
Official URL: https://doi.org/10.21769/BioProtoc.4763

Abstract

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.

Item Type: Journal Article
Publication: Bio-protocol
Publisher: Bio-protocol LLC
Additional Information: The copyright for this article belongs to the author.
Keywords: Co-purification; RNA–protein complex; ToxIN; Toxin–antitoxin system; Type III TA system
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 01 Aug 2023 05:12
Last Modified: 01 Aug 2023 05:12
URI: https://eprints.iisc.ac.in/id/eprint/82729

Actions (login required)

View Item View Item