N(pro) fusion technology: On-column complementation to improve efficiency in biopharmaceutical production

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• N(pro) fusion technology is an efficient system for overexpressing proteins and peptides in Escherichia coli.
• Existing methods face limitations in tag size and recyclability, impacting overall productivity and cost-effectiveness.

Purpose of the Study:

• To develop a novel functional complementation system by splitting the N(pro) autoprotease into two fragments.
• To reduce the size of the expression tag and enhance the efficiency and recyclability of the N(pro) fusion system.
• To demonstrate the applicability of the system for various model proteins and its potential for large-scale recombinant protein production.

Main Methods:

• Splitting the N(pro) autoprotease into two functional fragments to create a complementation system.
• Testing the system's efficacy with model proteins of varying sizes, including a peptide, MCP-1, and lysozyme.
• Immobilizing the N-terminal fragment to a solid support to enable fragment recycling.

Main Results:

• The split N(pro) system successfully restored auto-proteolytic activity upon complementation of the fragments.
• The expression tag size was significantly reduced by 66%, from 168 to 58 amino acids.
• The system demonstrated functionality with diverse model proteins and enabled efficient recycling of the immobilized N-terminal fragment.
• Productivity was shown to increase up to 4-fold for batch refolding and potentially more for on-column refolding strategies.

Conclusions:

• The developed N(pro) complementation system offers a more efficient and cost-effective approach for recombinant protein and peptide production.
• The reduced tag size and recyclability of the immobilized fragment significantly enhance overall productivity.
• This technology holds substantial potential for improving yields and reducing costs in large-scale biopharmaceutical manufacturing.