Production of native recombinant proteins using a novel split intein affinity technology.

Area of Science:

• Biotechnology
• Protein Chemistry
• Affinity Purification

Background:

• Affinity tags aid recombinant protein purification but often require removal due to biological interference and immunogenicity.
• Traditional tag removal necessitates expensive proteases, posing a challenge for cost-effective protein production.
• Split intein systems offer a protease-free alternative by enabling self-cleavage after protein capture.

Purpose of the Study:

• To evaluate the applications and advantages of the new Cytiva™ ProteinSelect™ split intein system.
• To demonstrate the system's capability for large-scale protein purification and resin reusability.
• To assess the optimization requirements for efficient protein expression and recovery using this system.

Main Methods:

• Utilized the Cytiva™ ProteinSelect™ system for capturing SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager.
• Investigated resin regeneration and sanitization using sodium hydroxide for multiple cycles.
• Performed binding studies with IL-1b and IFNAR-1 ECD to assess long-term binding capacity and reusability.

Main Results:

• Successfully captured and scaled up production of target proteins (SARS-CoV-2 spike protein RBD, Bispecific T Cell Engager) 10-fold.
• Demonstrated robust resin binding capacity (10-20 g/L) and retention of capacity after up to 50 regeneration/sanitization cycles.
• Confirmed retained binding capacity for target proteins after multiple sanitization cycles, highlighting resin durability.

Conclusions:

• The Cytiva™ ProteinSelect™ system is a feasible and advantageous tool for academic and industrial protein purification.
• The system offers robust protein capture, efficient tag removal, and durable, reusable affinity resin.
• Optimization of protein N-terminal sequences and expression conditions is crucial for maximizing cleavage efficiency and recovery.