Process optimization for increased yield of surface-expressed protein in Escherichia coli

Area of Science:

• Microbiology
• Protein Engineering
• Biotechnology

Background:

• Autotransporter proteins facilitate Gram-negative bacterial protein export.
• Surface expression of recombinant proteins using autotransporters is hindered by proteolysis.
• SefA, a Salmonella enterica fimbrial subunit, shows vaccine potential but suffers from low surface expression due to degradation.

Purpose of the Study:

• To enhance surface expression yield of the model protein SefA.
• To minimize proteolysis of SefA during export.
• To optimize the bioprocess for increased full-length SefA surface display.

Main Methods:

• Utilized Design of Experiments (DoE) methodology for process optimization.
• Investigated the impact of cultivation temperature, pH, and inducer concentration on SefA expression.
• Focused on parameters influencing periplasmic protease activity.

Main Results:

• Achieved a 200% increase in total SefA surface expression yield.
• Increased the yield of full-length SefA by 300%.
• Demonstrated a 33% reduction in protein proteolysis through parameter modification.

Conclusions:

• Process design and optimization effectively enhance recombinant protein surface expression in Gram-negative bacteria.
• Optimized conditions significantly reduce proteolysis, leading to higher yields of full-length functional proteins.
• The optimized SefA expression system holds promise for vaccine development applications.