Refolding

Refolding is the process by which a protein that has been denatured or unfolded regains its native three-dimensional structure and function. In biotechnology, refolding most often refers to the in vitro restoration of proteins produced in forms that require denaturation, such as inclusion bodies formed during bacterial expression. Proteins can be unfolded with chaotropic agents, detergents, or extreme pH, and refolded by gradually removing the denaturant or by dilution, dialysis, or on-column methods.

Successful refolding depends on multiple factors, including the protein’s length and complexity, the presence of disulfide bonds, temperature and pH, protein concentration, and the redox environment for disulfide formation. Aggregation is a major challenge, so conditions are optimized to minimize aggregation while promoting correct folding. Common strategies include stepwise or gradual removal of denaturant, the use of folding aids such as L-arginine, glycerol, or osmolytes, and redox shuffling with reduced and oxidized glutathione. In vitro refolding can also involve adding molecular chaperones or chaperone-like components to assist folding, or co-expressing folding catalysts in vivo.

On-column refolding and pulse-refolding are alternative approaches designed to limit aggregation by controlling how folding occurs during or after a purification step. After refolding, proteins are evaluated for correct structure and activity using functional assays, spectroscopy, or structural methods. Refolding is essential in producing many recombinant proteins at industrial scale; challenges remain to improve yield, activity, and stability. When refolding fails, other strategies include expressing the protein in a soluble form, using different host systems, or redesigning the protein sequence.