High-Throughput IgG Reformatting and Expression Using Hybrid Secretion Signals and InTag Positive Selection...

Area of Science:

• Molecular Biology
• Immunology
• Biotechnology

Background:

• Established protocols exist for high-throughput IgG reformatting and expression using phage-displayed antibody fragments.
• Existing methods can be cumbersome when dealing with diverse N-terminal sequences in phage libraries, complicating PCR amplification.
• The 5' PCR primer binding site is a critical point of difficulty in current antibody reformatting workflows.

Purpose of the Study:

• To overcome limitations in high-throughput IgG reformatting workflows.
• To simplify the PCR amplification step for diverse phage library clones.
• To enable efficient cloning of Fab fragments into IgG expression vectors.

Main Methods:

• Investigated the replacement of the C-terminal 7 amino acids of the human antibody VH1 secretion signal.
• Utilized bacterial ompA or pelB signal sequences to create hybrid signal sequences.
• Tested the efficacy of hybrid signals in driving IgG expression in Expi293F cells.

Main Results:

• Hybrid signal sequences, combining human VH1 with bacterial ompA or pelB signals, successfully drive strong IgG expression.
• The use of hybrid signals simplifies the amplification and cloning process.
• A single 5' PCR primer targeting the bacterial secretion signal is sufficient for cloning Fab fragments.

Conclusions:

• Hybrid secretion signals offer a significant simplification of the IgG reformatting workflow.
• This method dramatically streamlines the conversion of phage-displayed Fab fragments into expressible IgGs.
• The simplified workflow enhances the efficiency of antibody engineering and expression.