Novel column-based protein refolding strategy using dye-ligand affinity chromatography based on macroporous...

Area of Science:

• Biomaterials Science
• Protein Chemistry
• Chromatography

Background:

• Protein misfolding is a significant challenge in biotechnology and therapeutics.
• Existing refolding methods often suffer from low efficiency and protein dilution.

Purpose of the Study:

• To develop a novel, efficient, and scalable column-based protein refolding strategy.
• To utilize dye-ligand affinity chromatography (DLAC) with a macroporous biomaterial matrix.

Main Methods:

• A chitosan-silica (CS-silica) biomaterial was functionalized with Cibacron Blue F3GA (CBF) dye-ligand.
• The resulting CBF-CS-silica adsorbent was used in a column to rapidly remove denaturant (urea) from denatured catalase.
• Protein refolding was optimized by monitoring tryptophan fluorescence and enzyme activity.

Main Results:

• The developed DLAC strategy successfully refolded denatured catalase.
• The chromatographic refolding achieved a 20-fold increase in protein concentration compared to dilution refolding.
• Yields were comparable to or better than those obtained with dilution refolding.

Conclusions:

• Column-based protein refolding using DLAC with a porous biomaterial matrix is a promising technique.
• This method offers significant advantages in protein concentration and refolding efficiency.
• The strategy has potential for industrial applications in protein refolding.