Engineered sigma factors increase full-length antibody expression in Escherichia coli

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• Escherichia coli (E. coli) offers advantages for antibody production, including rapid growth and low cost.
• Current E. coli systems yield low titers (<5 mg/L) in shake flasks, limiting their use for research-grade antibody production.
• Enhancing antibody expression in E. coli is crucial for efficient antibody discovery programs.

Purpose of the Study:

• To increase the expression levels of full-length antibodies in E. coli using global transcriptional machinery engineering (gTME).
• To develop a high-throughput screening method for identifying E. coli mutants with enhanced antibody production.
• To enable rapid production of research-grade antibodies for early-stage characterization.

Main Methods:

• Generated a library of E. coli mutants with modifications in the global sigma factor RpoD.
• Employed bacterial antibody display (BAD) for high-throughput screening of mutants.
• Optimized expression by including chaperone co-expression.

Main Results:

• Identified specific RpoD mutants that significantly enhance full-length antibody titers.
• Achieved antibody titers up to 130.7 ± 6.6 mg/L in shake flask cultures.
• Demonstrated the feasibility of producing research-grade antibodies for characterization.

Conclusions:

• gTME is an effective strategy for improving antibody expression in E. coli.
• The developed method allows for rapid and cost-effective production of antibodies.
• This approach supports accelerated antibody discovery and characterization in early development phases.