Native Ubiquitin Purification via Affinity Tag Removal
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Solution Overview
Problem
Conventional methods for purifying ubiquitin often result in contamination with acid-resistant or heat-resistant proteins and can denature ubiquitin, limiting its purity and effectiveness.
Innovation Solution
A method involving the expression of affinity tag-linked ubiquitin in a strain, followed by affinity tag-based purification, dialysis, and chromatography without additional protease cleavage or heat treatment, allowing for the separation and concentration of high-purity ubiquitin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If acid or heat treatment is used to purify ubiquitin, then ubiquitin can be separated from other proteins, but other acid-resistant or heat-resistant proteins may contaminate the ubiquitin and the purity is restricted
Solution Approach 1:
The patent extracts the ubiquitin protein from the cell lysate using affinity chromatography with a specific affinity column that selectively binds ubiquitin, thereby separating it from other proteins including acid-resistant and heat-resistant proteins. This extraction method achieves high purity without the contamination issues of conventional acid/heat methods.
Solution Approach 2:
The patent uses an affinity column as an intermediary medium that specifically interacts with ubiquitin through affinity binding. This intermediary allows selective capture of ubiquitin from the complex protein mixture, enabling high-purity separation without harsh conditions.
2Manufacturing precision
If acid or heat treatment is used to purify ubiquitin, then ubiquitin can be precipitated and separated, but ubiquitin might be affected or denatured by acid or heat treatment
Solution Approach 1:
The patent replaces the mechanical/physical harsh methods (acid treatment and heat treatment) with a biochemical affinity-based separation system. The affinity column uses specific molecular interactions to separate ubiquitin, eliminating the need for denaturing conditions that could damage protein structure.
Solution Approach 2:
The patent changes the separation parameter from physical conditions (pH, temperature) to biochemical properties (affinity binding). By using affinity chromatography, the separation is based on specific molecular recognition rather than harsh physical parameters, preserving protein stability.
3Manufacturing precision
If affinity tag-based purification is used, then ubiquitin can be selectively purified, but the method leaves remnants of protease cleavage site in the target protein making it difficult to use for native ubiquitin purification
Solution Approach 1:
The patent extracts and removes the affinity tag sequence from the expressed protein using proteolytic enzymes. This step eliminates the tag and its associated protease cleavage sites, yielding native ubiquitin without any tag remnants or cleavage site residues that would compromise structural integrity.
Solution Approach 2:
The patent discards the affinity tag as a temporary tool used during purification, then removes it through proteolytic cleavage. The tag serves its purpose during purification but is subsequently eliminated to recover native, tag-free ubiquitin with intact structure.
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 method achieves high-purity ubiquitin purification, exceeding 99% purity without denaturing the protein, overcoming the limitations of conventional techniques.
Implementation Method 1
performing affinity tag-based first purification using the lysate obtained in Step (b)
Implementation Method 2
dialyzing the first purified product and separating the affinity tag from the ubiquitin
Implementation Method 3
performing second purification through chromatography using the dialyzed product obtained in Step (d)
Data Source
AI summary
The present invention relates to a method of purifying native ubiquitin without acid or heat treatment. The method presented here is designed to overcome limitations of acid or heat based ubiquitin purification in two different points of view. First, it decreases a chance of mixing other proteins resistant to acids or heat. Second, it includes no harsh condition, which might denature the ubiquitin. As a result, the purification of native ubiquitin becomes possible. The ubiquitin obtained herein is expected to be used for various purposes in technical fields.