Arginine Stabilizes Polymerase Storage Without Detergents
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Solution Overview
Problem
Thermostable polymerases used in applications like PCR face storage stability challenges due to potential protein denaturation caused by detergents, and existing solutions do not effectively maintain enzyme activity over long periods without detergents.
Innovation Solution
A storage solution comprising a polymerase with a 5'-3' exonuclease domain fused to a family B polymerase, conjugated to a DNA binding domain, dissolved in a solution with free arginine or its derivatives and a cryoprotectant, without detergents, which enhances stability and maintains activity at various temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If detergents (non-ionic or zwitterionic) are added to storage buffer, then polymerase storage stability is improved, but risk of protein denaturation increases
Solution Approach 1:
The patent removes detergents from the storage buffer formulation entirely, extracting the harmful component while maintaining stability through alternative means (arginine and cryoprotectants). This resolves the contradiction by eliminating the source of potential denaturation while achieving stability through a different mechanism.
Solution Approach 2:
The patent changes the chemical parameters of the storage buffer by substituting detergent-based stabilization with arginine and cryoprotectant-based stabilization. This parameter change allows achieving storage stability without introducing the harmful denaturation effect associated with detergents.
2Stability of the object's composition
If detergents are used in storage solution, then polymerase stability is enhanced, but enzyme activity is reduced over time
Solution Approach 1:
The patent extracts detergents from the storage formulation to prevent the gradual loss of enzyme activity that occurs with detergent-containing buffers, while maintaining stability through arginine and cryoprotectants.
Solution Approach 2:
The patent uses a composite stabilization system combining arginine and cryoprotectants to achieve both stability and activity preservation, replacing the single-component detergent-based approach that fails to maintain long-term activity.
3Duration of action of stationary object
If storage temperature is increased to extend shelf-life, then storage duration is improved, but polymerase denaturation accelerates
Solution Approach 1:
The patent applies beforehand cushioning by including arginine and cryoprotectants in the storage buffer before storage, which protect the polymerase from thermal denaturation during extended storage at elevated temperatures, thereby extending shelf-life without sacrificing stability.
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 solution significantly extends the storage shelf-life of polymerases by 9-fold, maintaining enzymatic activity and stability even at elevated temperatures, outperforming detergent-based solutions.
Implementation Method 1
arginine improves polymerase storage stability
Implementation Method 2
5% or more cryoprotectant
Data Source
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AI summary
Liquid storage solutions comprising polymerases are provided. In some embodiments, provided herein are liquid storage solution for storing a polynucleotide manipulating enzyme (e.g., a polymerase or other enzyme as described herein). In some embodiments, the solution comprises, the enzyme (e.g., polymerase orrestriction enzyme); free arginine or a salt thereof or an arginine derivative (for example, but not limited to arginine ethyl ester, argininainide dihydrochloride) or a salt thereof; and 5% or more cryoprotectant.