Important parameters affecting efficiency of protein refolding by reversed micelles

Area of Science:

• Biochemistry
• Protein chemistry
• Biophysical chemistry

Background:

• Inclusion bodies are protein aggregates that often require refolding for therapeutic or research use.
• Traditional refolding methods using reversed micelles involve liquid-liquid extraction, which can be inefficient.
• Developing alternative, efficient methods for protein refolding is crucial for biotechnology.

Purpose of the Study:

• To develop and optimize a novel solid-liquid extraction method for refolding denatured Ribonuclease A (RNase A) using reversed micelles.
• To investigate the effects of operational parameters on protein solubilization, renaturation, and recovery.
• To demonstrate the efficacy of this method for producing active refolded proteins, even at high concentrations.

Main Methods:

• Utilized reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane for protein refolding.
• Employed a solid-liquid extraction technique as an alternative to conventional liquid-liquid extraction.
• Investigated the impact of AOT concentration, water content (W(o)), and pH on protein solubilization and renaturation.
• Used glutathione as a redox reagent for renaturation and acetone for protein recovery via precipitation.

Main Results:

• Optimized conditions (high AOT concentration, high W(o), high pH) facilitated efficient solubilization of solid denatured protein aggregates.
• Complete renaturation of RNase A was achieved by adjusting redox reagent composition, even at high protein concentrations that typically cause aggregation.
• Renaturation rates were enhanced by optimizing W(o) and pH within the reversed micellar solution.
• Acetone precipitation effectively recovered renatured RNase A from the reversed micellar solution with minimal loss of enzymatic activity.

Conclusions:

• A novel solid-liquid extraction method using reversed micelles provides an effective alternative for refolding denatured proteins.
• The method allows for efficient solubilization, renaturation, and recovery of active RNase A, overcoming challenges associated with high protein concentrations.
• Optimization of reversed micellar parameters (AOT concentration, W(o), pH) is key to successful protein refolding and recovery.