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Updated: Aug 13, 2025
14:25
Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
Published on: December 12, 2017
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Intensification of Inclusion Body Processing via Temperature-Based Refolding.
Ru Shen Wong1, Nurul Nadia Mohamad Alias1, Eugene Boon Beng Ong2
1Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia.
Methods in Molecular Biology (Clifton, N.J.)
|January 19, 2023
Summary
A novel temperature-based refolding method efficiently recovers soluble core streptavidin from inclusion bodies. This approach uses high temperatures and low denaturant concentrations, simplifying purification and reducing buffer waste.
Area of Science:
- Biochemistry
- Protein Chemistry
- Biotechnology
Background:
- Inclusion bodies (IB) are aggregates of misfolded proteins formed during recombinant expression.
- Conventional methods to recover soluble proteins from IB involve high denaturant concentrations, leading to dilute products and buffer inefficiency.
- Efficient refolding of proteins from IB is crucial for biotechnology and therapeutic applications.
Purpose of the Study:
- To describe a temperature-based refolding approach for core streptavidin (cSAV) recovery from IB.
- To demonstrate the intensification and efficiency of this novel refolding method.
- To achieve high-purity refolded cSAV without column purification.
Main Methods:
- Solubilization of IB using a low concentration of guanidine hydrochloride (0.5 M GdnHCl) combined with high temperature (95 °C).
- Concurrent denaturant removal/reduction and refolding in a favorable chemical environment.
- Characterization of refolded cSAV using biotin-binding assay, SDS-PAGE, and RP-HPLC for purity assessment.
Main Results:
- The temperature-based approach achieved protein refolding intensification by eliminating concentration steps and reducing buffer volumes.
- High-temperature treatment during solubilization aided in denaturing and aggregating host-cell proteins, facilitating their removal via centrifugation.
- High-purity refolded cSAV was obtained, bypassing the need for traditional column purification.
Conclusions:
- The temperature-based refolding approach offers an intensified and buffer-efficient alternative to conventional methods for recovering soluble proteins from IB.
- This method simplifies downstream processing, yielding highly pure refolded proteins.
- The approach holds significant potential for improving recombinant protein production in various expression systems.
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