A high-throughput genetic system for assessing the inhibition of proteins: identification of antibiotic resistance and virulence targets and their cognate inhibitors in Salmonella.

Anal Biochem

Pre-harvest Food Safety and Enteric Disease Research Unit, National Animal Disease Center, Agricultural Research Service/USDA, 2300 Dayton Road, Box 70, Ames, IA 50010, USA.

Published: November 2002

This study describes the development of a high-throughput genetic system for producing oligopeptides that can be used to identify molecular interactions leading to inhibition of specific proteins. Using a pathogenic bacteria model, we screened a library of clones expressing intracellular oligopeptides in order to identify inhibitors of proteins involved in antibiotic resistance and virulence. This method involved transforming the pathogen with an oligopeptide-encoding plasmid library, constructed using polymerase chain reaction and an oligonucleotide template designed to produce random oligopeptides composed of 2-16 amino acids, and high-throughput screening for phenotype alterations in the pathogen. A subsequent complementation phase enabled the identification of the full-length bacterial protein inhibited by the oligopeptide. Using this method we were able to identify oligopeptides that inhibit virulence and/or drug resistance in Salmonella, Shigella, and Escherichia coli; specific virulence and/or drug resistance proteins of Salmonella, Shigella, and E. coli that are sensitive to inhibition; and putative oligopeptide-binding sites on the inhibited proteins. This system is versatile and can be extended to other pathogens for analogous studies and it can be modified for used in eukaryotic models for identifying protein interactions that can be targeted for inhibition. Additionally, this system can be used for identifying protein domains involved in any biomolecular interaction.

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Source
http://dx.doi.org/10.1016/s0003-2697(02)00251-8DOI Listing

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