A yEGFP‐based reporter system for high‐throughput yeast two‐hybrid assay by flow cytometry

Abstract

The yeast two-hybrid (Y2H) assay is a widely used method to study protein-protein interactions, a major objective in postgenome research. Despite the tremendous utility of the Y2H assay, several issues including accuracy, speed, automation, and cost-effectiveness limit its application in high-throughput analysis. We have created an improved Y2H assay reporter system by integrating the codon-optimized yeast enhanced green fluorescent protein (yEGFP) gene into the ADE2 locus of the AH109 yeast strain and evaluated reporter expression using the strong and weak triggers via flow cytometry. We also performed flow cytometry-based Y2H assays in liquid cultures as well as in a cDNA library screening. We have shown that yEGFP, but not EGFP, is a sensitive Y2H reporter for flow cytometric detection. We also show that this yEGFP-based Y2H could be easily adapted to high-throughput format without conventional agar plating. Moreover, our data demonstrate that this system can be used for cDNA library screening. We have developed sensitive and efficient flow cytometry-based Y2H assay system that is well suited for large-scale protein-protein interaction identification and characterization. When compared with the conventional plate and filter membrane-based nutrient and colorimetric analysis, our flow cytometric assay offers convenient, quantitative, and faster reporter analysis compatible with existing liquid handling robots. ' 2008International Society for Analytical Cytology Key terms flow cytometry; yEGFP; EGFP; yeast two-hybrid; high throughput; cDNA library screening; fluorescence; protein-protein interaction PROTEIN-protein interactions are fundamental to virtually every aspect of cellular function. To understand the protein-protein interaction network on a large scale, the genome-wide protein linkage mapping or ''interactome'' analyses have therefore become a major objective in the field of proteomics (1). Although a number of promising technologies have been developed to analyze and monitor protein-protein interactions in vitro and in vivo, and the need for confirmation by independent methods is recognized (2), only the yeast two-hybrid (Y2H) and mass spectrometrybased affinity copurification approaches have been proven robust and versatile enough for systematic large-scale analysis (3-6).The Y2H system is a genetic screen wherein the interaction between two proteins of interest is detected via the functional reconstitution of the distinct DNA binding and activation domains of a transcription factor and the subsequent activation of reporter expression controlled by the transcription factor (7). Briefly, bait and prey protein pairs are fused to distinct DNA binding and activation domains of a transcription factor (e.g., GAL4). The interaction of bait and prey protein pairs brings the DNA binding and activation domains into close proximity, forming an active transcription factor complex, which binds the promoter and triggers the expression of reporter gene(s) under the control of that promoter. The Y2H assay do...

Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

“…Recent studies have applied this tool widely, for example in genome-wide approaches to analyze messenger RNA (mRNA) abundance910, transcriptional regulation11, protein abundance or localization31213, and protein–protein interactions1415. In many cases, these studies used GFP or other chromophoric proteins for the facile tracking and monitoring of target proteins.…”

High throughput flow cytometry based yeast two‐hybrid array approach for large‐scale analysis of protein‐protein interactions

“…PGBKT7-VP16 (BD-VP16) was made by cloning the VP16 activation domain into the EcoRI / SalI sites of the pGBKT7 vector as a positive control for the self-activators. The AH109-yEGFP reporter strain was constructed as described (7). The Y187-yEGFP reporter strain was constructed as follows: the GAL1 promoter was amplified from the pYM_N22 plasmid (EUROSCARF) with OJC-201 (5’-AGgtcgacGAGCTCTAGTACGGATTAG-3’) and OJC-202 (5’-gggttaattaaATCCGGGGTTTTTTCTCCTT-3’) primers.…”

“…In this study, we sought to employ HT flow cytometry for large-scale analysis of PPIs through the use of the GFP reporter based Y2H system which we recently established (7). Flow cytometry is a versatile high speed cell analysis method that is playing key roles in proteomics and systems biology efforts (23).…”

Application of flow cytometry to industrial microbial bioprocesses

“…), intracellular pH, enzymatic activity and membrane integrity [275]. The gene encoding green fluorescent protein (GFP) and its variations has become an important reporter in the study of gene expression dynamics within living bacteria, yeasts or eukaryotic cultures [97][98][99]285,286]. By monitoring these parameters over time, data can be used to construct detailed kinetic profiles of the cell populations, providing a deeper understanding of the kinetics of these processes.…”