Mutant DHFR Destabilizing Domains for Tunable Protein Stabilization
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Solution Overview
Problem
Existing methods for regulating protein function in mammalian cells are limited, often resulting in high basal levels of transgene expression and lack specificity, and there is a need for reliable and tunable methods to conditionally stabilize proteins for efficient gene therapy applications.
Innovation Solution
Mutant polypeptides derived from Escherichia coli dihydrofolate reductase (DHFR) with specific mutations at positions W133, F153, R12, N18, M42, Y100, D122, P126, and D127 are fused to a polypeptide of interest, allowing conditional modulation of activity through binding with a DHFR inhibitor like trimethoprim, stabilizing the fusion protein only in the presence of the inhibitor.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transcriptional level gene expression targeting methods (Cre/Iox, tet/dox systems) are used, then gene expression can be modulated, but relatively high basal levels of transgene expression occur when not induced
Solution Approach 1:
The invention extracts the destabilizing function from the protein of interest by fusing it to a destabilizing domain (DD). The DD contains specific amino acid residues that make the fusion protein unstable and subject to degradation by the proteasome, thereby removing the problematic basal expression at the source rather than attempting to control it transcriptionally.
Solution Approach 2:
The invention introduces a stabilizing ligand as an intermediary molecule that binds to the fusion protein and prevents its degradation. This ligand acts as a mediator between the destabilizing domain and the protein of interest, allowing conditional stabilization only when the ligand is present, thereby achieving tight control with minimal basal expression.
2Speed
If inhibitors or activators of particular proteins are used, then protein activity can be modulated with speed and reversibility, but these molecules are often promiscuous and affect several proteins rather than a specific protein
Solution Approach 1:
The invention applies local quality by creating a highly specific interaction interface between the destabilizing domain and its stabilizing ligand. The DD is engineered with specific amino acid residues that create a unique binding pocket, ensuring that only the intended ligand can stabilize the fusion protein. This localized specificity prevents promiscuous effects on other proteins while maintaining fast and reversible control.
3Adaptability or versatility
If methods for regulating protein function directly are used, then control over protein activity can be achieved, but these methods are limited especially in mammalian cells
Solution Approach 1:
The invention achieves universality by creating a modular destabilizing domain that can be fused to any protein of interest in mammalian cells. The DD-proteasome degradation pathway is a universal cellular mechanism that works across different cell types, allowing the same DD-Ligand system to be applied universally for controlling various proteins in mammalian systems without requiring cell-type-specific modifications.
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 enables precise control over protein activity, allowing for efficient shutdown of protein function in the absence of the inhibitor and dose-dependent activation in response to ligand administration, suitable for personalized gene therapy treatments.
Implementation Method 1
Mutant polypeptides derived from Escherichia coli dihydrofolate reductase (DHFR) with specific mutations at positions W133, F153, R12, N18, M42, Y100, D122, P126, and D127 are fused to a polypeptide of interest, allowing conditional modulation of activity through binding with a DHFR inhibitor like trimethoprim, stabilizing the fusion protein only in the presence of the inhibitor
Data Source
AI summary
The present disclosure relates to mutant polypeptides derived from Escherichia coli dihydrofolate reductase (DHFR) which can be fused to a polypeptide of interest for efficient conditional modulation of its activity. Also disclosed are polynucleotides encoding such mutant polypeptides, vectors comprising such polynucleotides, and the use of such polypeptides, polynucleotides and vectors for treating a disorder.