A simple plasmid-based transient gene expression method using High Five cells.

Area of Science:

• Biotechnology
• Insect cell culture
• Recombinant protein production

Background:

• The High Five (H5) cell line from Trichoplusia ni is a key host for recombinant protein production via baculovirus expression vector system (BEVS).
• BEVS requires time-consuming viral vector construction and optimization for protein expression.

Purpose of the Study:

• To establish a simple, rapid, and efficient polyethylenimine (PEI)-based transient gene expression (TGE) process for recombinant protein production in suspension-adapted H5 cells.
• To optimize TGE conditions and evaluate its performance as an alternative to BEVS.

Main Methods:

• Optimization of promoter and enhancer combinations for high protein yield using enhanced green fluorescent protein (EGFP) and a human tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc).
• Selection of an expression vector with the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer.
• Transfection of H5 cells at 2×10(6) cells/mL using direct addition of DNA and PEI.

Main Results:

• Achieved 90% transfection efficiency with EGFP as a reporter.
• Obtained volumetric TNFR-Fc yields exceeding 150μg/mL within 4 days post-transfection.
• Demonstrated reproducibility and scalability up to 300mL.

Conclusions:

• PEI-based TGE is a simple, efficient, and rapid method for recombinant protein production in H5 cells.
• This plasmid-based transient transfection process serves as a viable alternative to BEVS for H5 cell-based protein expression.