Stabilized, long-term expression of heterodimeric proteins from tricistronic mRNA

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Long-term stability of recombinant protein overexpression in mammalian cells is a significant challenge, especially when foreign gene products are toxic to producer cells.
• Existing methods often struggle with maintaining stable expression over extended periods.

Purpose of the Study:

• To develop and validate a vector system for the stable expression of heterodimeric recombinant proteins in mammalian cells.
• To address the issue of long-term expression instability caused by toxic protein products.

Main Methods:

• Designed a tricistronic expression vector system where two recombinant cDNAs and a puromycin-resistance gene are transcribed as a single unit.
• Utilized internal ribosome entry sites (IRES) to mediate efficient translation of internal cistrons.
• Demonstrated the system's efficacy using a heterodimeric antibody fusion protein expressed in BHK-21 cells.

Main Results:

• Achieved stable, long-term expression of a heterodimeric antibody fusion protein.
• Showcased that translational coupling to a selectable marker stabilizes expression under continuous selection pressure.
• Validated the vector system's effectiveness in BHK-21 cells.

Conclusions:

• The developed tricistronic vector system enables the generation of producer cell lines with unlimited long-term stability for complex heterodimeric proteins.
• This system offers a fast and straightforward method for constructing expression plasmids for challenging protein overexpression applications.