Lysozyme refolding with immobilized GroEL column chromatography

Area of Science:

• Biochemistry
• Protein Chemistry
• Chromatography

Background:

• Protein misfolding is a significant challenge in biotechnology.
• Molecular chaperones like GroEL are crucial for proper protein folding.
• Efficient refolding techniques are needed for therapeutic protein production.

Purpose of the Study:

• To investigate the refolding of denatured-reduced lysozyme using immobilized GroEL chromatography.
• To optimize operating parameters for enhanced protein refolding yield and specific activity.
• To evaluate the refolding productivity compared to existing methods.

Main Methods:

• Utilized immobilized molecular chaperonin GroEL in a chromatography column.
• Studied the effects of denaturant concentration, elution flow-rate, and substrate protein loading.
• Analyzed the recovery yield of lysozyme mass and its specific activity.

Main Results:

• Lysozyme mass recovery remained high (90-100%) across tested parameters.
• Specific activity of refolded lysozyme was significantly influenced by operating conditions.
• An optimal denaturant concentration of approximately 1 M yielded up to 97% specific activity.
• Achieved a refolding productivity of 54 mg/gel/h, over four times higher than previous methods.

Conclusions:

• Immobilized GroEL chromatography offers a highly efficient system for protein refolding.
• The method demonstrates a significant advantage in producing high throughput for protein refolding operations.
• This approach holds promise for large-scale production of correctly folded proteins.