Multi-host expression system for recombinant production of challenging proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Expression

Background:

• Recombinant production of complex eukaryotic proteins requires extensive screening for optimal constructs and expression hosts to ensure high yield and proper folding.
• Host selection significantly impacts protein yield, quality, and overall production costs.

Purpose of the Study:

• To present a novel, rapid, multi-host expression system (pFlp-Bac-to-Mam) for efficient recombinant protein production.
• To demonstrate the system's versatility in optimizing expression strategies for diverse protein classes.

Main Methods:

• Developed a single donor vector, pFlp-Bac-to-Mam, for multi-host expression.
• Utilized HEK293-6E cells for transient transfection in serum-free suspension culture.
• Employed the baculoviral expression vector system and stable CHO production cell lines (via Flp-mediated cassette exchange) for large-scale protein production.
• Evaluated expression of three model proteins: fluorescent protein, single-chain-Fv-hIgG1Fc fusion, and Toll-like receptor extracellular domain.

Main Results:

• The pFlp-Bac-to-Mam system facilitated high-level expression (mg/L) of challenging proteins.
• Demonstrated successful intracellular expression, secretion, and production of homogeneous protein samples.
• Comparative studies identified optimal expression strategies for different protein types.

Conclusions:

• The pFlp-Bac-to-Mam system provides a fast and adaptable platform for optimizing recombinant protein expression across various hosts.
• Enables efficient production of high-quality, properly folded eukaryotic proteins for structural and functional studies.