Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System

Area of Science:

• Biotechnology
• Molecular Biology
• Virology

Background:

• The Semliki Forest Virus (SFV) system is a powerful tool for recombinant protein expression.
• Scaling up protein production presents challenges, including cell aggregation and maintaining optimal conditions.

Purpose of the Study:

• To scale up the Semliki Forest Virus (SFV) expression system for recombinant protein production to bioreactor scale.
• To optimize expression conditions for high yields of the human 5-HT(3) receptor.

Main Methods:

• Subcloning the human 5-HT(3) receptor gene into the SFV expression plasmid pSFV1.
• Utilizing a Vibromix agitation system to manage BHK cell suspension culture in a 11.5 L bioreactor.
• Infection of cell cultures with recombinant virus and subsequent protein purification via a hexa-His tag.

Main Results:

• Achieved an expression level of 3 million functional 5-HT(3) receptors per cell 24 hours post-infection.
• Optimized conditions, including pH 7.3 control, increased yields to 8 million receptors per cell (a twofold increase).
• Demonstrated successful single-step purification with 51% yield.

Conclusions:

• The SFV expression system is effective for rapid, large-scale recombinant protein production.
• Optimized bioreactor conditions significantly enhance protein yield and efficiency.
• This scalable system offers an attractive method for producing substantial quantities of recombinant proteins quickly.