Enhanced periplasmic expression of high affinity humanized scFv against Hepatitis B surface antigen by codon...

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Recombinant antibody production in prokaryotes faces challenges like low expression levels.
• Rare codons in native antibody genes can hinder efficient protein synthesis in E. coli.
• Previous work generated a Hepatitis B surface antigen-specific scFv (5S) with low E. coli expression.

Purpose of the Study:

• To improve the production of a functional Hepatitis B surface antigen-specific single-chain variable fragment (scFv) in Escherichia coli.
• To investigate the impact of codon optimization on scFv expression and function.
• To overcome low expression levels caused by codon bias in the native scFv gene.

Main Methods:

• Designed a synthetic 5S-scFv gene using E. coli preferred codons.
• Cloned the codon-optimized gene into a T5 expression vector with a C-terminal His-tag.
• Expressed the scFv in E. coli periplasm and purified it using immobilized metal affinity chromatography (IMAC).
• Analyzed expression levels, protein solubility, and antigen-binding activity.

Main Results:

• Codon optimization led to a >100-fold increase in soluble periplasmic scFv expression.
• The purified scFv demonstrated stability and retained antigen-binding affinity and epitope specificity.
• Secondary structure analysis suggested mRNA secondary structure played a minor role in the enhanced expression.

Conclusions:

• Codon optimization is a highly effective strategy for boosting recombinant scFv production in E. coli.
• The engineered scFv is functional, stable, and retains its specific binding to Hepatitis B surface antigen.
• This approach facilitates the development of prokaryotic expression systems for antibody-based therapeutics.