muNS Fusion Protein Microspheres for Protein Interaction Detection
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Solution Overview
Problem
Existing systems for detecting protein interactions using the muNS protein from mammalian and avian reoviruses face issues such as incorrect protein folding, interference with inclusion formation, and unsuitable intracellular localization, leading to reduced effectiveness in protein detection and purification.
Innovation Solution
A fusion protein is developed comprising a polypeptide based on the minimal region of the Orthoreovirus muNS protein, modified with a sortase recognition sequence, allowing for the addition of molecules at the C-terminal end to enhance inclusion formation and facilitate post-translational modifications, enabling the formation of nanospheres and microspheres.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the target protein is fused to the C-terminal region of muNS to form cytoplasmic inclusions, then the detection of protein interactions is improved, but the target protein may fold incorrectly and lose the capacity to interact with its ligands
Solution Approach 1:
The invention divides the muNS protein into two functional segments: the N-terminal region (amino acids 1-447) that forms the inclusion body, and the C-terminal region (amino acids 448-721) that can be fused with the target protein. This segmentation allows the N-terminal region to self-assemble into inclusions while the C-terminal fusion partner maintains its proper folding and interaction capacity, resolving the contradiction between inclusion formation and protein functionality.
Solution Approach 2:
The invention introduces a linker peptide as an intermediary between the muNS protein and the target protein. This linker serves as a flexible connector that allows the target protein to fold correctly while still being recruited into the muNS inclusion bodies, thereby maintaining both the detection capability and the protein's native function.
2Measurement precision
If the target protein is fused to the C-terminal region of muNS, then protein interaction detection is enhanced, but the protein may interfere with inclusion formation and generate amorphous aggregates
Solution Approach 1:
By segmenting muNS into N-terminal (1-447) and C-terminal (448-721) regions, the invention ensures that the N-terminal region maintains its self-assembling capability to form ordered inclusion bodies, while the C-terminal region serves as a fusion partner that does not interfere with the structural integrity of the inclusion formation process.
Solution Approach 2:
The invention applies local quality by assigning different functional properties to different regions of the fusion protein. The N-terminal region is optimized for inclusion body formation with specific self-assembling properties, while the C-terminal region is designed to maintain flexibility and compatibility with various target proteins, allowing each region to perform its specialized function without interfering with the other.
3Adaptability or versatility
If the C-terminal end of muNS is modified to add molecules, then the versatility of the system is improved, but the capacity to form inclusions may be affected
Solution Approach 1:
The invention segments muNS such that the N-terminal region (1-447) is dedicated to inclusion body formation, while the C-terminal region (448-721) is designed to accommodate various modifications including fusion with target proteins, epitope tags, and other molecular additions. This segmentation allows unlimited versatility in C-terminal modifications without compromising the inclusion formation capacity of the N-terminal region.
Solution Approach 2:
The C-terminal region of muNS (amino acids 448-721) is designed as a universal platform that can accommodate multiple different target proteins and modifications. This universal design allows the same N-terminal inclusion-forming core to work with diverse C-terminal partners, enhancing the system's versatility while maintaining stable inclusion formation.
Data Source
Figure 1a~2c
Figure 3~4
Figure 5A~5D
AI summary
The invention relates to a fusion protein encoding a polypeptide based on the minimal region of the Orthoreovirus muNS protein capable of forming microspheres and/or nanospheres, modified to permit the addition of polypeptides at the C-terminal.