Recombinant Protein Refolding Using Chaotropic Agents and Oxygen
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Solution Overview
Problem
Bacterial expression systems, such as E. coli, face challenges in properly refolding recombinant proteins due to the lack of cellular machinery for protein folding, resulting in misfolded and inactive proteins, and existing methods for refolding are not always efficient or sufficient for large-scale production.
Innovation Solution
A method involving the use of buffered solutions with chaotropic agents and reducing agents, along with the addition of air or oxygen, to solubilize and refold recombinant proteins from prokaryotic host cells, specifically using solutions with urea, arginine, DTT, and cysteine at controlled pH and temperature conditions to achieve proper folding and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If bacterial expression systems are used to produce recombinant proteins, then production cost is reduced and cell density is increased, but the proteins are misfolded and biologically inactive due to lack of cellular machinery for proper refolding
Solution Approach 1:
The invention changes the chemical parameters of the refolding environment by using buffered solutions with specific pH ranges (pH 7-9) and controlled concentrations of chaotropic agents (0.1-2 M) and reducing agents (0.1-10 mM). These parameter changes create optimal conditions for proper protein refolding, resolving the contradiction between cost-effective bacterial production and correct protein folding.
Solution Approach 2:
The invention introduces chemical intermediaries (chaotropic agents like urea or guanidine hydrochloride, and reducing agents like DTT or beta-mercaptoethanol) that mediate the refolding process. These intermediaries temporarily disrupt misfolded structures and facilitate correct folding, bridging the gap between bacterial production capabilities and eukaryotic-level protein folding requirements.
2Adaptability or versatility
If typical refolding methods using denaturant dissolution followed by dilution are used, then the process is universally applicable, but the methods do not eliminate misfolded intermediates or provide sufficient amounts of properly folded product
Solution Approach 1:
The invention modifies the refolding parameters by maintaining specific pH ranges (7-9) and using controlled dilution factors, which prevents the formation of misfolded intermediates while maintaining broad applicability across different protein types. This resolves the contradiction between universal applicability and folding accuracy.
Solution Approach 2:
The invention incorporates monitoring steps to assess refolding progress and adjust conditions accordingly, ensuring sufficient production of properly folded protein while maintaining method versatility. This feedback mechanism allows optimization for each specific protein while preserving the general applicability of the approach.
3Productivity
If refolding is performed at higher protein concentrations, then productivity is improved, but association through hydrophobic interaction increases leading to misfolded multimers and dimers
Solution Approach 1:
The invention changes the chemical environment parameters by adding chaotropic agents and reducing agents that counteract hydrophobic interactions at higher concentrations. This allows refolding to proceed efficiently at productive protein concentrations without forming misfolded multimers, resolving the contradiction between productivity and folding correctness.
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 effectively recovers and purifies biologically active, properly folded recombinant proteins, enhancing the efficiency and scalability of protein production by improving the refolding process and achieving high yields of correctly folded proteins.
Implementation Method 1
solubilizing and the soluble protein refolded into the native state
Implementation Method 2
two or more reducing agents and addition of air or oxygen for such a time and under such conditions that refolding of the recombinant protein occurs
Implementation Method 3
two or more reducing agents and addition of air or oxygen for such a time and under such conditions that refolding of the recombinant protein occurs
Implementation Method 4
refolding said solubilized protein in a second buffered solution, pH >9 but ≤ 11
Data Source
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AI summary
Processes are provided for recovering and purifying refolded recombinant proteins produced in heterologous host cells, which includes the step of refolding the protein in a high pH buffer.