Recombinant Cell System for Protein-Protein Interaction Transduction
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Solution Overview
Problem
Current biosensors primarily generate detectable signals that require manual readout and interpretation, with limited progress in transducing signals into downstream biological activities such as gene expression, particularly for protein-protein interactions like GPCR signaling, which is crucial for drug discovery and cell therapies.
Innovation Solution
A recombinant cell system that utilizes histidine kinase variants with retained DHp and CA domains to facilitate protein-protein interactions, enabling the phosphorylation of response regulatory proteins and subsequent modulation of gene expression, allowing for the transduction of protein-protein interactions into specific biological responses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If conventional biosensors are used to detect protein-protein interactions, then detectable signals are generated, but manual readout and interpretation are required, limiting automation
Solution Approach 1:
The patent introduces an intermediary transcription factor that acts as a mediator between the protein-protein interaction and the gene expression output. The transcription factor is released upon proteolytic cleavage when the target protein interacts with the sensor, and this freed transcription factor then activates gene expression. This intermediary mechanism enables automatic transduction of the biological signal into a measurable output without manual intervention.
Solution Approach 2:
The patent replaces manual readout and interpretation mechanisms with a biological automation system. Instead of requiring human operators to read and interpret signals, the system uses proteolytic cleavage to automatically release a transcription factor that directly activates gene expression, substituting mechanical/manual operations with a self-executing biological cascade.
2Adaptability or versatility
If protein-protein interactions are transduced to detectable signals, then interaction detection is enabled, but transduction to downstream biological activities like gene expression is limited
Solution Approach 1:
The patent creates a universal transduction platform where the sensor-protease fusion protein can detect various protein-protein interactions and the released transcription factor can activate multiple different gene expression outputs. This multi-functional design allows the same core mechanism to be applied to different targets and outputs, enhancing versatility while maintaining a relatively simple modular structure.
Solution Approach 2:
The transcription factor serves as a universal intermediary that decouples the detection mechanism from the output mechanism. The sensor releases the transcription factor upon detecting any protein-protein interaction, and this freed transcription factor can then activate various downstream genes, enabling versatile signal transduction to different biological activities through a common intermediary.
3Measurement precision
If high dynamic range detection is achieved for low protein-protein interactions, then detection sensitivity is improved, but system complexity increases
Solution Approach 1:
The patent segments the detection system into distinct functional modules: a sensor domain for binding, a protease domain for signal amplification through cleavage, and a transcription factor for output. This segmentation allows each component to be optimized independently for its specific function, achieving high sensitivity through the coordinated action of specialized modules rather than a single complex system.
Solution Approach 2:
The patent implements continuous signal amplification through the catalytic nature of the protease and transcription factor. A single protein-protein interaction event triggers proteolytic cleavage that releases the transcription factor, which can then continuously activate gene expression until the transcription factor is degraded or sequestered. This continuous biological action maintains high signal levels and improves detection sensitivity for low-abundance interactions.
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 approach provides a high dynamic range and reversibility, enabling the detection of low protein-protein interactions and multiplexing capabilities, facilitating the development of advanced screening assays and therapeutic applications by directly linking protein-protein interactions to gene expression.
Implementation Method 1
the structural basis of HK autophosphorylation is the existence of two distinct HK dimer conformations. Upon ligand binding, the CA domain and the bound ATP get into close proximity of the histidine of the DHp, enabling phosphoryl transfer.
Implementation Method 2
the CA domain and the bound ATP get into close proximity of the histidine of the DHp, enabling phosphoryl transfer.
Implementation Method 3
a cognate response regulator (RR) binds to the phosphorylated DHp domain, the phosphate is transferred from the histidine to one aspartate in the receiver domain of the RR, and the phosphorylated RR binds to its target promoters and regulates gene expression.
Data Source
AI summary
The present invention relates to a cell comprising a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, wherein said first variant does not comprise a transmembrane domain, a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, wherein said second variant does not comprise a transmembrane domain, and a third nucleic acid sequence encoding a response regulatory protein specifically phosphorylatable by said DHp domain of said first or said second variant. The present invention further relates to uses of the cell of the invention.