Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins

Area of Science:

• Microbiology
• Biochemistry
• Protein Engineering

Background:

• Colicins are antimicrobial proteins from *Escherichia coli* with potential as antibiotic alternatives.
• Cell-free protein synthesis (CFPS) is a viable platform for producing toxic proteins like colicins.
• Previous studies showed CFPS-produced colicins can eliminate antibiotic-tolerant persister cells, but some colicins exhibited poor solubility and activity.

Purpose of the Study:

• To optimize colicin production using cell-free protein synthesis (CFPS) for improved solubility and cell-killing activity.
• To enhance the production and efficacy of specific colicins, such as colicin M and colicin E3.
• To demonstrate the rapid synthesis and sustained activity of colicins via CFPS.

Main Methods:

• Production of colicin M in chaperone-enriched *E. coli* extracts to improve solubility.
• CFPS of colicin E3 with the addition or co-expression of its cognate immunity protein (E3 IP) to enhance cytotoxicity.
• Monitoring colicin E1 production yield and cytotoxicity over time during CFPS.

Main Results:

• Colicin M solubility was increased from 16% to nearly 100% with enhanced cell-killing activity.
• The E3 immunity protein addition/co-expression improved colicin E3 cytotoxicity, suggesting a role beyond host protection.
• Colicin E1 reached maximum production yield within 3 hours of CFPS incubation and maintained high cytotoxicity for up to 20 hours.

Conclusions:

• CFPS platform allows for efficient optimization of colicin production.
• Strategies like chaperone enrichment and immunity protein co-expression significantly enhance colicin solubility and activity.
• Rapid and effective synthesis of active colicins is achievable using optimized CFPS, supporting their development as antimicrobial agents.