I’m currently attempting to express a recombinant mouse IgG antibody in HEK293F cells, but I’ve encountered challenges with low or undetectable expression levels. I would greatly appreciate any expert insights to help identify the issues and guide improvements. Below is a detailed description of my construct and protocol:

De Novo Sequencing

I performed de novo sequencing on a mouse IgG antibody and obtained its full amino acid sequence. I compared the variable and constant regions to IMGT references:

• The variable regions (VH and VL) show good alignment with IMGT germline sequences but contain many mutations, as expected.
• The constant regions, including the Fc domain, also show some mutations compared to IMGT references.

Plasmid Design & Expression Strategy

Full plasmid info:

https://en.vectorbuilder.com/vector/VB250423-1922bnk.html

Key features of the plasmid:

• Single plasmid co-expression system for both heavy chain (HC) and light chain (LC).
• Codon-optimized for mammalian expression (specifically HEK293).
• IL-2 signal peptides included of both HC and LC to promote secretion.
• EF1A promoter drives HC expression; CMV promoter drives LC expression. This was based on prior data showing CMV is stronger than EF1A in HEK293, aiming to balance HC and LC expression.
• GFP reporter included to monitor transfection efficiency.

Transfection Protocol (Using PEI)

Problem Observed

• Transfection efficiency was moderate (~30–40% GFP+).
• No clear antibody bands were seen on SDS-PAGE under reducing conditions (very faint HC or LC bands).
• After concentration and buffer exchange, precipitation was observed. It’s unclear whether this is the antibody or other protein aggregates.

Key Questions

Thank you all!!