Control of aggregation in protein refolding: the temperature-leap tactic

Area of Science:

• Biochemistry
• Protein Folding Kinetics

Background:

• Protein aggregation is a major challenge during refolding.
• Bovine carbonic anhydrase II (CAII) aggregation kinetics were previously uncharacterized.

Purpose of the Study:

• To investigate the refolding kinetics of bovine carbonic anhydrase II (CAII).
• To explore the efficacy of a "temperature leap" strategy to enhance CAII refolding yield.
• To understand the role of intermediate states in aggregation and refolding.

Main Methods:

• Studied CAII renaturation kinetics at various temperatures (4°C to 36°C) and high protein concentrations.
• Utilized guanidinium chloride dilution for protein unfolding.
• Performed kinetic simulations to model folding pathways and aggregation.
• Employed a "temperature leap" approach, varying initial incubation temperatures before warming.

Main Results:

• High CAII concentrations (4 mg/mL) led to rapid aggregation, reducing active enzyme yield.
• Refolding at 4°C dramatically reduced aggregation, yielding 37% activity.
• Warming refolded CAII from 4°C to 36°C rapidly increased yield to 95%.
• Initial incubation temperatures between 4°C and 12°C followed by warming yielded >90% activity.

Conclusions:

• The "temperature leap" strategy effectively minimizes aggregation and maximizes refolding yield for CAII.
• Hydrophobic aggregation is suppressed at lower temperatures, facilitating productive folding pathways.
• This approach holds potential for optimizing the refolding of other therapeutic proteins.