DOI: 10.6084/m9.figshare.29948877
title: An <i>E. coli</i> two-hybrid system to investigate human protein-protein interactions

description: The LexA-<i>E. coli</i> two-hybrid (LexA-E2H) system was initially developed to study interactions between microbial proteins in an <i>Escherichia coli</i> (<i>E. coli)</i> 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. The LexA-<i>E. coli</i> 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. <b>Method Summary</b>: 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 <i>E. coli</i> employing colorimetric colony screening on MacConkey agar and quantitative β-galactosidase assays to identify and measure PPIs. This study investigates the interaction between GRP78, an endoplasmic reticulum chaperone, and the cleaved-Par-4 (cl-Par-4) tumor suppressor, using the LexA-E2H system.The LexA-E2H (<i>E. coli</i> two-hybrid) system is a cost-effective, efficient alternative to traditional methods for studying human protein-protein interactions (PPIs).LexA-E2H avoids the need for culturing eukaryotic cells and bypasses interference from eukaryotic proteins. It is ideal for resource-limited laboratories, and is complementary to standard techniques.GRP78 and cl-Par-4 were sub-cloned into LexA vectors.MacConkey agar was used for colorimetric colony screening to detect the interaction.β-galactosidase assays provided quantitative assessment of the interaction.Successful construction and expression of GRP78 and cl-Par-4 fusion proteins were verified using Western blot analysis.Colorimetric screening on MacConkey agar distinguished between pale white-pink colonies (+ PPI) and red colonies (− PPI), confirming interaction between GRP78 and cl-Par-4.β-galactosidase assays quantitated the strength of the interaction between GRP78 and cl-Par-4 in this context.The LexA-E2H system offers a simple, cost-effective alternative to yeast two-hybrid (Y2H) and co-immunoprecipitation/mass spectrometry (coIP/MS) methods.It reduces false positives/negatives common in Y2H due to interactions mediated by eukaryotic homologs.Suitable for academic teaching laboratories, it expands accessibility to PPI studies for students and educators in resource-limited environments.This study provides the first direct empirical evidence of the PPI between GRP78 and cl-Par-4, and suggests that post-translational modifications (PTM) are not required for this interaction. These results demonstrate the utility of the LexA-E2H system for human PPI research.The LexA-E2H system, coupled with β-galactosidase assays, effectively detects and quantitates the interaction between cl-Par-4 and GRP78.The LexA-E2H system is straightforward to implement, making it an excellent alternative for PPI analysis in research and teaching environments.LexA-E2H complements traditional yeast-based and other PPI techniques by providing a non-eukaryotic environment, thus avoiding interference from endogenous mammalian proteins and PTMs. This study investigates the interaction between GRP78, an endoplasmic reticulum chaperone, and the cleaved-Par-4 (cl-Par-4) tumor suppressor, using the LexA-E2H system. The LexA-E2H (<i>E. coli</i> two-hybrid) system is a cost-effective, efficient alternative to traditional methods for studying human protein-protein interactions (PPIs). LexA-E2H avoids the need for culturing eukaryotic cells and bypasses interference from eukaryotic proteins. It is ideal for resource-limited laboratories, and is complementary to standard techniques. GRP78 and cl-Par-4 were sub-cloned into LexA vectors. MacConkey agar was used for colorimetric colony screening to detect the interaction. β-galactosidase assays provided quantitative assessment of the interaction. Successful construction and expression of GRP78 and cl-Par-4 fusion proteins were verified using Western blot analysis. Colorimetric screening on MacConkey agar distinguished between pale white-pink colonies (+ PPI) and red colonies (− PPI), confirming interaction between GRP78 and cl-Par-4. β-galactosidase assays quantitated the strength of the interaction between GRP78 and cl-Par-4 in this context. The LexA-E2H system offers a simple, cost-effective alternative to yeast two-hybrid (Y2H) and co-immunoprecipitation/mass spectrometry (coIP/MS) methods. It reduces false positives/negatives common in Y2H due to interactions mediated by eukaryotic homologs. Suitable for academic teaching laboratories, it expands accessibility to PPI studies for students and educators in resource-limited environments. This study provides the first direct empirical evidence of the PPI between GRP78 and cl-Par-4, and suggests that post-translational modifications (PTM) are not required for this interaction. These results demonstrate the utility of the LexA-E2H system for human PPI research. The LexA-E2H system, coupled with β-galactosidase assays, effectively detects and quantitates the interaction between cl-Par-4 and GRP78. The LexA-E2H system is straightforward to implement, making it an excellent alternative for PPI analysis in research and teaching environments. LexA-E2H complements traditional yeast-based and other PPI techniques by providing a non-eukaryotic environment, thus avoiding interference from endogenous mammalian proteins and PTMs.