An E. coli Two-Hybrid System to Investigate Human Protein-Protein Interactions

Authors

Rebecca M. Richardson, Old Dominion UniversityFollow
Sarah M. Ho, Old Dominion UniversityFollow
Lane Tong, Old Dominion UniversityFollow
Morgan G. Daniels, Old Dominion UniversityFollow
Steven M. Pascal, Old Dominion UniversityFollow

Document Type

Article

Publication Date

2025

DOI

10.1080/07366205.2025.2542028

Publication Title

Biotechniques

Volume

77

Issue

7-8

Pages

283-295

Abstract

Abstract

The LexA-E. coli two-hybrid (LexA-E2H) system was initially developed to study interactions between microbial proteins in an Escherichia coli (E. coli) environment. We here demonstrate its utility for studying mammalian protein interactions. Specifically, this study uses LexA-E2H to provide the first direct and quantitative validation of Glucose Regulated Protein 78 (GRP78) binding to the cleaved-Prostate Apoptosis Response 4 (cl-Par-4) tumor suppressor. Furthermore, the results establish that this interaction does not require phosphorylation of either protein. MacConkey agar was used for initial detection of the interaction through colorimetric colony screening, distinguishing pale white-pink colonies (+ interaction) from red colonies (- interaction). This was followed by β-galactosidase assays for quantitative assessment. These results demonstrate the potential of the LexA-E2H system to advance human protein-protein interaction research. LexA-E2H is simple to implement, avoiding the need to culture eukaryotic cells, and bypassing interference from eukaryotic proteins. This system is ideal for laboratories with limited resources and complements conventional eukaryotic methods.

Multidisciplinary Abstract

The LexA-E. coli two-hybrid (LexA-E2H) system offers an affordable and efficient way to study interactions between human proteins. It is straightforward to use, eliminating the need to grow complex eukaryotic cells and avoiding interference from their proteins. This makes it especially useful for labs with limited resources. This study used the LexA-E2H system to show that Glucose Regulated Protein 78 (GRP78) binds to cleaved-Prostate Apoptosis Response 4 (cl-Par-4). The system uses a special agar for initial detection of the interaction and a subsequent enzyme assay for quantitative analysis. These findings demonstrate the potential of the LexA-E2H system to advance research on human protein interactions.

Method Summary:

LexA-E2H system provides a cost-effective, efficient alternative for studying human protein-protein interactions (PPI). This method uses a straightforward genetic screening process in E. coli employing colorimetric colony screening on MacConkey agar and quantitative β-galactosidase assays to identify and measure PPIs.

Rights

© 2025 The Authors.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the accepted manuscript in a repository by the authors or with their consent.

Original Publication Citation

Richardson, R. M., Ho, S. M., Tong, L., Daniels, M. G., & Pascal, S. M. (2025). An E. coli two-hybrid system to investigate human protein-protein interactions. Biotechniques, 77(7-8), 283-295. https://doi.org/10.1080/07366205.2025.2542028

Repository Citation

Richardson, Rebecca M.; Ho, Sarah M.; Tong, Lane; Daniels, Morgan G.; and Pascal, Steven M., "An E. coli Two-Hybrid System to Investigate Human Protein-Protein Interactions" (2025). Chemistry & Biochemistry Faculty Publications. 361.
https://digitalcommons.odu.edu/chemistry_fac_pubs/361