Soluble Recombinant COQ5 Protein Purification via Low-Concentration Detergent
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Solution Overview
Problem
Current methods for purifying recombinant coenzyme Q5 protein expressed in E. coli often result in denatured or aggregated forms, leading to low yields and difficulties in antibody production, as denaturing conditions are required to solubilize the protein, causing issues with protein stability and purification efficiency.
Innovation Solution
Treating bacterial cell lysate with a low concentration of ionic detergent, such as SDS, allows for the dissociation of MTS-truncated human COQ5 protein from bacterial membranes, enabling purification under native conditions without aggregation or precipitation, and subsequent purification using Ni-NTA beads without urea.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If denaturing conditions (8 M urea) are used to purify recombinant COQ5 protein, then the protein can be solubilized and purified, but the protein becomes aggregated or precipitated upon removal of urea, reducing yield and requiring lengthy buffer-exchange processes
Solution Approach 1:
The patent changes the concentration parameter of ionic detergent from high (denaturing conditions) to low (0.01-0.5% w/v), enabling protein solubilization under native conditions without requiring subsequent urea removal steps. This parameter change resolves the contradiction by maintaining solubility while avoiding the time-consuming buffer exchange process.
Solution Approach 2:
The patent introduces ionic detergent as an intermediary substance that facilitates protein dissociation from inclusion bodies under mild conditions. This intermediary enables solubilization without requiring extreme denaturing agents like 8 M urea, thus avoiding the aggregation problem upon urea removal and eliminating lengthy buffer-exchange processes.
2Quantity of substance
If denaturing conditions are used to purify recombinant COQ5 protein, then the protein can be purified, but the procedures become more complicated and additional purification steps are required
Solution Approach 1:
The patent simplifies the purification procedure by changing the ionic detergent concentration parameter to a low range (0.01-0.5% w/v), which allows protein solubilization under native conditions. This eliminates the need for multiple complex steps including urea removal and refolding, thereby reducing overall procedure complexity while maintaining protein solubility.
3Quantity of substance
If denaturing conditions are used to purify recombinant COQ5 protein, then the protein can be purified, but the protein yield is reduced due to aggregation and precipitation
Solution Approach 1:
The patent changes the ionic detergent concentration parameter to a low range (0.01-0.5% w/v), which solubilizes the protein under native conditions without causing aggregation or precipitation. This parameter optimization maximizes protein yield by preventing loss through aggregation while maintaining solubility throughout the purification process.
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 maintains the protein in a soluble form for extended periods, improving yield and stability, facilitating effective antibody production and affinity purification, and ensuring the protein remains usable for biochemical studies and pharmaceutical applications.
Implementation Method 1
treating the cell lysate with low concentration of ionic detergent... allows for the dissociation of MTS-truncated human COQ5 protein from bacterial membranes
Implementation Method 2
applying the supernatant to Ni-NTA beads under native condition to purify the recombinant soluble COQ5 protein
Data Source
AI summary
The present invention relates to a method of producing and purifying soluble recombinant coenzyme Q5 homolog (COQ5) protein, which is expressed in soluble form by Escherichia coli (E. coli), under native conditions. The method is characterized by pre-treating bacterial lysate with low concentration of ionic detergent, such as sodium dodecyl sulfate (SDS), before purification; and the purifying method is performed under native condition without using urea to avoid the problems of requiring lengthy processes to remove urea in purified protein solution or re-aggregation and precipitation of protein after removal of urea.