Degron Fusion Tags for Graded Protein Expression Control
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Solution Overview
Problem
Existing methods struggle to achieve precise and graded expression of both endogenous and exogenous proteins, which is crucial for understanding their physiological functions and conducting in vitro investigations, especially in mammalian cells, where factors like vector, promoter, mRNA stability, and protein stability complicate protein expression regulation.
Innovation Solution
A degron system is introduced, comprising a short peptide sequence with at least 70% sequence identity to SEQ ID NO: 1, fused to proteins to regulate their expression by promoting proteasomal degradation, allowing for fine-tuned protein levels through wild-type and mutant degron sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used to express proteins in mammalian cells, then protein expression can be achieved, but precise and graded expression levels cannot be controlled
Solution Approach 1:
The invention segments the protein of interest into two parts: a stable version and an unstable version tagged with a degron sequence. By controlling the expression ratio between these two versions, precise graded expression levels can be achieved. The unstable tagged protein acts as a regulated component that can be selectively degraded, allowing fine-tuned control over the functional protein level in the cell.
Solution Approach 2:
The degron sequence acts as an intermediary element that mediates between the stable and unstable protein versions. This intermediary degron tag directs the unstable protein for proteasomal degradation, creating a controllable mechanism to regulate the net protein expression level. The intermediary element enables precise control without requiring complex external regulation systems.
2Ease of operation
If essential genes are deleted or knocked out to study their function, then physiological function investigation is simplified, but cell viability is lost
Solution Approach 1:
Instead of completely deleting essential genes, the invention applies partial action by expressing reduced levels of the essential gene product through the unstable tagged version. This allows the gene to be partially knocked down to investigate its function while maintaining sufficient expression levels to preserve cell viability. The controlled reduction enables functional studies without complete loss of essential functions.
3Adaptability or versatility
If multiple factors (vector, promoter, mRNA stability, protein stability) are optimized to control protein expression, then expression levels can be adjusted, but the system becomes complex and difficult to regulate
Solution Approach 1:
The invention extracts the stability control element from the complex regulation system by isolating it into a separate degron sequence that can be independently applied to any protein of interest. Instead of optimizing multiple factors simultaneously, the stability characteristic is extracted as a modular tag that can be added to proteins to confer controlled instability. This simplifies the regulation system while maintaining versatility across different proteins and expression contexts.
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 degron system enables controlled and varied expression levels of proteins, including GFP, RLuc, and mtDsRed, by targeting them for degradation, providing a robust method to study protein function and essential gene investigations.
Implementation Method 1
A system is provided to regulate the expression of a protein by fusion with a degron sequence
Data Source
AI summary
The present disclosure provides a degron comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1 for regulating the expression of a protein. The present disclosure also provides a fusion protein comprising a polypeptide of interest and the degron. Further, the present disclosure relates to a method of producing the fusion protein and a method of regulating the expression of a polypeptide of interest.