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

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT3 receptor was chosen. The gene for the receptor was su bcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latt er to a reactor volume of 11.5 l. A Vibromix(TM) agitation system was chose n to overcome aggregation problems of BHK cells in suspension. In the proce ss, cells were first grown to a density of 10(6) cells/ml, the medium was t hen exchanged with fresh medium and the culture was infected with the recom binant virus at an estimated multiplicity of infection of 30. 24 h post inf ection we measured an expression level of 3 million functional 5-HT3 recept ors per cell. For harvesting, the cells were pelleted by centrifugation. Th e receptor protein was purified in a single step (Hovius et al., 1998) by e xploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per ce ll, when the pH of a suspension culture was controlled at pH 7.3. Rapid vir us generation and protein production, high protein yields as well as succes sful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. A fter optimisation of the expression conditions (in particular by setting th e pH at 7.3), yields were increased twofold.