Fluorescent Two-Hybrid Assay for Real-Time Protein Interaction Detection
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Solution Overview
Problem
Current methods for detecting protein-protein interactions in mammalian cells are laborious, error-prone, and often require costly instrumentation or suffer from false positives due to the lack of stable expression of fluorescently labeled bait proteins and irreversible complementation, making them unsuitable for real-time detection.
Innovation Solution
An in vitro method involving the expression of fusion proteins in eukaryotic cells, where one protein accumulates in the nucleus and binds to GFP, another is fluorescently labeled, and a third has a distinct fluorescence wavelength, allowing for real-time detection of protein-protein interactions through co-localization of fluorescence emissions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If fluorescence-based methods (FRET or BiFC) are used for in-cell visualization of protein-protein interactions, then real-time detection capability is improved, but device complexity and cost increase due to costly instrumentation requirements
Solution Approach 1:
The patent uses fluorescent proteins (GFP and RFP) as optical copies or markers that bind to proteins of interest, allowing visualization of protein-protein interactions through fluorescence co-localization rather than requiring complex FRET instrumentation. The fluorescent tags serve as simplified proxies that can be detected with standard microscopy equipment
Solution Approach 2:
The patent employs stable expression of fluorescently labeled bait proteins in mammalian cells, eliminating the need for costly and complex FRET instrumentation. This approach uses affordable fluorescent markers and standard microscopy to achieve real-time detection, replacing expensive equipment with simpler, more accessible tools
2Ease of manufacture
If BiFC method is used for protein-protein interaction detection, then visualization capability is improved, but reliability deteriorates due to irreversible complementation and slow maturation preventing real-time detection
Solution Approach 1:
Instead of using irreversible complementation as in BiFC, the patent employs reversible binding interactions between fluorescent proteins and their targets. The stable expression system allows dynamic, reversible association that enables real-time monitoring of protein interactions without the irreversible trapping inherent in BiFC
Solution Approach 2:
The patent implements a dynamic system where fluorescently labeled bait proteins are stably expressed in mammalian cells, allowing continuous, reversible interaction with prey proteins. This dynamic approach enables real-time detection of interaction changes, unlike the static, irreversible BiFC complementation
3Productivity
If yeast two-hybrid system is used for protein-protein interaction screening, then high-throughput capability is improved, but reliability worsens due to absence of cellular factors and posttranslational modifications
Solution Approach 1:
The patent uses mammalian cells that naturally provide all required cellular factors, chaperones, and posttranslational modification enzymes. The system is self-sufficient, eliminating the need to supplement with external factors or risk false negatives due to missing cellular components
Solution Approach 2:
The patent transitions from yeast to mammalian cells, changing the cellular environment parameters to better match human protein interaction contexts. This includes changing the species-specific cellular machinery, posttranslational modification capabilities, and overall cellular physiology to improve reliability of interaction detection
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables direct visualization of protein-protein interactions in real-time without the need for advanced technical expertise or costly instrumentation, providing a reliable and high-throughput capable assay for studying protein interactions in living cells.
Implementation Method 1
detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction
Data Source
Figure 1a~1c
Figure 2a~2b
Figure 3a~3d
AI summary
The present invention relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell or interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus of the cell; and (ii) a (poly)peptide specifically binding to GFP; (b) expressing in the same cell a second fusion protein comprising (i) GFP; and (ii) a bait (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP; and (ii) a prey (poly)peptide; and (d) detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. The invention also relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait (poly)peptide (b) expressing in the same cell a second fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first fusion protein; and (ii) a prey (poly)peptide and (c) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. Furthermore, the present invention relates to methods for identifying a compound modulating the interaction of two (poly)peptides and methods of determining the relative strength of the interaction of two proteins with a third protein.