Untitled

Abstract

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-HT(3) receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A Vibromix(TM) agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 10(6) cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT(3) receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.

Semliki Forest Virus-Based Expression of Recombinant GPCRs

“…For this purpose, suspension cultures of BHK and CHO cells are grown in serumfree DHI medium, a mixture of DME, Ham F12, and IMDM media (1:1:1) supplemented with 5 mM glutamine, 5 g/L glucose, 5 mg/L insulin (Sigma, Buch, Switzerland), 6 mg/L transferrin, 0.25% (v/v) Primatone RL (Sheffield Products/Quest International, Norwich, NY), 0.01% (v/v) Synperonic F68 (Serva, Heidelberg, Germany), 20 mM selenite, 20 μM ethanol amine, and 2.5 mM β-mercaptoethanol (Schlaeger, 1996). To obtain singlecell suspension cultures, a Vibromix™ device (Gerber & Pfenninger, Switzerland) with two stainless steel plates (each containing 20 conical holes) of 5.5 cm diameter, fixed on a vibrating shaft (vibration: 50 Hz, amplitude 1.5 mm) can be applied (Blasey et al, 2000). Spinner flask (Bellco, Inotech AG, Dottikon, Switzerland) cultures are aerated at 95-105 rpm at 37°C using approximately 70% of the recommended working volume.…”

“…In the case of application of fermenters, a better control of growth condition parameters (pH, oxygen, and CO 2 ) can be established. For instance, when the serotonin 5-HT3 receptor was expressed in a bioreactor at 11.5-L scale optimal production was achieved by adjusting the pH to 6.9 for the infection phase and then 2 h postinfection increasing the pH to 7.4 (Blasey et al, 2000). As large-scale production in suspension cultures generally requires large quantities of virus stocks, this can be achieved by combining cells from several electroporations for virus production in larger culture flasks.…”

“…The mouse serotonin 5-HT3 receptor (ion channel) was expressed in bioreactors up to 11.5 L volume resulting in yields of up to 20 mg receptor (Blasey et al, 2000). The engineering of a histidine-tag at the C-terminus of the 5-HT3 receptor allowed determination of the oligomeric state of the receptor by gel filtration, secondary structure determination by circular dichroism and cryo-EM single particle imaging.…”

Transient Gene Expression in Mammalian Cells: Promises and Challenges for Medical Biotechnology

“…Alphavirus vectors have been used to generate secreted, membrane-associated, and intracellular proteins with volumetric yields up to 20 mg/L (60). In particular, the large-scale production of recombinant G protein-coupled receptors (GPCRs) has been performed with SFV expression vectors in suspension cultures of mammalian cells in bioreactors and spinner flasks (61,62).…”