Auxin-Degron System Kit for Efficient Protein Degradation
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Solution Overview
Problem
The membrane permeability of auxins or auxin analogs is impaired by barrier functions in eggshells or the blood-brain barrier, limiting the efficiency of protein degradation in non-plant-derived eukaryotic cells using the auxin-degron system.
Innovation Solution
An auxin-degron system kit that includes a mutant TIR1 family protein with a mutation at the auxin-binding site, an auxin analog with improved membrane permeability, and a degradation tag that includes a partial Aux/IAA family protein, enabling efficient protein degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If an auxin or auxin analog is used to induce protein degradation in the auxin-degron system, then the degradation efficiency of the target protein is improved, but the membrane permeability is impaired due to barrier functions in eggshells or the blood-brain barrier
Solution Approach 1:
The patent introduces a cell-permeable auxin analog as an intermediary substance that can cross biological membranes (eggshells, blood-brain barrier) and activate the TIR1 protein inside cells. This mediator bypasses the permeability barrier while maintaining the degradation function, resolving the contradiction between improving degradation efficiency and overcoming membrane permeability impairment.
Solution Approach 2:
The patent modifies the chemical structure of the auxin molecule to create an analog with improved pharmacological properties. By changing parameters such as molecular weight, lipophilicity, and chemical groups (e.g., adding acetoxymethyl ester groups), the auxin analog achieves both high degradation efficiency and improved membrane permeability, simultaneously resolving both aspects of the contradiction.
2Productivity
If a high concentration of auxin (100 μM or higher) is used to induce degradation, then the degradation effect is achieved, but toxic effects on multicellular animals occur
Solution Approach 1:
The patent optimizes the concentration parameter by using cell-permeable auxin analogs that are more potent and selective. This allows achieving effective degradation at much lower concentrations (nanomolar to low micromolar range) compared to traditional auxin, thereby maintaining degradation effect while eliminating toxic effects on multicellular animals.
Solution Approach 2:
The patent employs a temporary, controlled degradation mechanism where the auxin analog is used only when needed to trigger protein degradation, then rapidly metabolized or excreted. This disposable approach avoids cumulative toxicity while maintaining effective degradation during the active period, resolving the contradiction between achieving degradation effect and avoiding toxic effects.
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 achieves high-efficiency control of protein degradation with improved membrane permeability of the auxin analog, reducing the concentration required for effective protein removal and minimizing toxic effects.
Implementation Method 1
a first nucleic acid encoding a mutant TIR1 family protein that has a mutation at an auxin-binding site; an auxin analog having an affinity for the mutant TIR1 family protein
Implementation Method 2
binds to the degradation tag through the complex with the auxin analog, leading to degradation of the target protein
Data Source
AI summary
An auxin-degron system kit controlling degradation of a target protein in a non-plant-derived eukaryotic cell, in which the auxin-degron system kit includes a first nucleic acid encoding a mutant TIR1 family protein that has a mutation at an auxin-binding site; an auxin analog having an affinity for the mutant TIR1 family protein; and a second nucleic acid encoding a degradation tag that includes at least a partial Aux/IAA family protein and has an affinity for a complex of the mutant TIR1 family protein and the auxin analog.