A novel bicistronic vector inducing balanced alternative splicing for antibody production.

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• Recombinant monoclonal antibody production requires balanced expression of light (LC) and heavy (HC) chains.
• Existing bicistronic systems (IRES, 2A peptides) face challenges like imbalanced translation and residual peptide tags, impacting antibody quality.

Purpose of the Study:

• To develop a novel bicistronic expression platform, the cassette exon-associated (CAE) system, for efficient and stable recombinant antibody production.
• To overcome limitations of conventional bicistronic systems by avoiding imbalanced translation and peptide tags.

Main Methods:

• Developed the CAE system utilizing alternative splicing from the HEG1 gene to generate two mRNA isoforms from a single promoter.
• Constructed an optimized vector (pCAE3.31) with synthetic splice donor and acceptor sequences.
• Tested the CAE system with various promoters (chicken β-actin, CMV, EF-1α) in multiple mammalian cell lines.

Main Results:

• The CAE system efficiently expressed functional antibodies and Fab fragments without additional peptide sequences.
• Achieved an 8-fold higher antibody yield compared to IRES-based systems.
• Demonstrated compatibility with major mammalian promoters and expression in various cell lines.

Conclusions:

• The CAE system is a compact, versatile bicistronic strategy for expressing LC and HC, avoiding peptide fusions.
• Facilitates efficient production of recombinant antibodies and Fab fragments for screening and therapeutic applications.
• Represents an advancement over IRES and 2A peptide systems for antibody manufacturing.