Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries.

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• Antibody fragments are expressed in Escherichia coli and directed to the periplasm for proper folding and disulfide bond formation using N-terminal signal sequences.
• Periplasmic expression faces challenges like inefficient translocation and limited system capacity.
• Modulating signal sequence codon usage is a strategy to improve translocation and antibody fragment expression levels.

Purpose of the Study:

• To establish synonymous PelB signal sequence libraries by modulating codon usage in light and heavy chains of a Fab fragment.
• To screen these libraries for clones with improved expression profiles.
• To analyze the effects of codon usage and mRNA secondary structures on Fab fragment expression levels.

Main Methods:

• Created three synonymous PelB signal sequence libraries by mutating n-region, hydrophobic region, and c-region of light and heavy chains in a bicistronic expression vector.
• Screened libraries for clones exhibiting enhanced expression.
• Analyzed the impact of codon usage and mRNA secondary structures on expression levels.

Main Results:

• Improved clones were obtained from all three signal sequence libraries, with the n-region library being the most productive.
• Codon usage at the fifth leucine position of the light chain PelB signal sequence significantly affects Fab fragment expression.
• mRNA secondary structures in the translation initiation regions of both light and heavy chains influence expression levels.

Conclusions:

• Synonymous signal sequence libraries serve as valuable resources for identifying Fab fragments with enhanced expression.
• This study provides novel information regarding codon usage and its impact on antibody fragment expression in Escherichia coli.