Optimisation of surface expression using the AIDA autotransporter.

Area of Science:

• Microbiology
• Biotechnology
• Protein Engineering

Background:

• Bacterial surface display is crucial for applications like live vaccines and biocatalysts.
• Understanding parameters for high-yield, high-display recombinant protein production is essential.
• Staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli (E. coli).

Purpose of the Study:

• To identify key parameters influencing recombinant protein production and surface display.
• To optimize conditions for high cell density and desired protein expression levels on the cell surface.
• To investigate the impact of strain design and medium composition on protein display.

Main Methods:

• Utilized an OmpT-negative E. coli mutant to prevent protein degradation.
• Optimized culture medium, identifying minimal medium with glucose as superior to LB-medium.
• Employed glucose-limited fed-batch and batch cultivation strategies to enhance cell productivity.
• Investigated the effect of repeated glucose additions in batch cultures.

Main Results:

• The OmpT-negative mutant demonstrated successful surface expression, unlike the wild type which showed degradation.
• Expression in the mutant resulted in a narrower distribution of surface-anchored protein.
• Minimal medium with glucose yielded over four times higher expression than LB-medium.
• High surface expression was maintained up to 18 g l-1 cell dry weight in fed-batch and batch cultures.
• Prolonged batch processing was limited by low oxygen levels.

Conclusions:

• Medium choice and strain design (preventing proteolytic cleavage) are critical for surface protein expression.
• Both fed-batch and batch processing are viable for high-yield surface display.
• Strain selection, particularly low acetic acid production, is important for prolonged batch cultivation.
• Optimized conditions lead to improved recombinant protein productivity and display consistency.