Avian Orthoreovirus muNS Intercoil Domain Fusion Protein Detection
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Solution Overview
Problem
Current systems for detecting protein interactions, such as the double hybrid system and those using mammalian reovirus muNS protein, face issues like mis-folding, interference with inclusion formation, and unsuitable intracellular location, limiting their effectiveness and versatility.
Innovation Solution
A fusion protein system is developed using a polypeptide of interest fused with specific domains of the avian Orthoreovirus muNS protein, specifically the Intercoil domain, which forms cytoplasmic inclusions, allowing for efficient detection of protein interactions without altering the inclusion formation and maintaining protein activity, and can be adapted for nuclear behavior.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the double hybrid system or mammalian reovirus muNS protein is used for detecting protein interactions, then protein interaction detection can be achieved, but mis-folding and interference with inclusion formation occur, limiting effectiveness
Solution Approach 1:
The patent divides the muNS protein into functional domains, specifically using the C-terminal domain (amino acids 471-635) which contains the inclusion formation capability. This segmentation allows the detection system to use only the necessary functional portion, reducing mis-folding risks while maintaining inclusion formation ability for protein interaction detection.
Solution Approach 2:
The patent extracts the essential inclusion-forming domain from the complete muNS protein. By using a truncated version (C-terminal domain) rather than the full-length protein, the system eliminates regions that may cause mis-folding or interference, while preserving the core functionality of inclusion formation and protein interaction detection.
2Adaptability or versatility
If existing protein interaction detection systems are used, then interaction detection is possible, but the intracellular location is unsuitable and versatility is limited
Solution Approach 1:
The patent creates a universal detection platform based on the C-terminal domain of muNS that can detect protein interactions in both cytoplasmic and nuclear compartments. The system is designed to work with various target proteins regardless of their subcellular location, achieving multi-functionality and broad versatility in protein interaction detection.
Solution Approach 2:
The C-terminal domain of muNS acts as an intermediary that bridges the detection of protein interactions in different cellular compartments. This mediator component enables the system to adapt to various intracellular locations by facilitating inclusion formation and protein recruitment without being restricted by specific subcellular positioning.
3Productivity
If multiple proteins are integrated into inclusions, then detection capability increases, but inclusion formation may be interfered with
Solution Approach 1:
The patent applies local quality by concentrating the inclusion formation function in the C-terminal domain of muNS, while other regions of the system are optimized for specific detection functions. This spatial functional differentiation allows multiple proteins to be integrated into inclusions without compromising the core inclusion formation mechanism, as each component operates in its optimized location.
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
The system effectively detects protein interactions with lower mis-folding risks, allows multiple proteins to be integrated into inclusions, and is suitable for nuclear protein interactions, providing a more versatile and accurate method compared to existing systems.
Implementation Method 1
a polypeptide of interest fused with specific domains of the avian Orthoreovirus muNS protein, specifically the Intercoil domain, which forms cytoplasmic inclusions
Data Source
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AI summary
The invention is based on the identification of the minimum region of the avian Orthoreovirus muNS protein which is capable of forming inclusions as well as on the identification of specific regions of the muNS protein showing capacity to associate with the inclusions formed by muNS. The identification of said regions allows developing methods for purifying recombinant polypeptides as well as methods for detecting the interaction between two polypeptides of interest.