Infoscience

Journal article

Expression of ligand-gated ion channels with the Semliki Forest virus expression system

Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system. The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT3 and SFV-P2x virus stocks. Infection of BHK, CHO, and RIN cells resulted in high-level expression of recombinant receptors. Satn. binding anal. indicated the presence of >3 * 106 5-HT3 receptors/cell. Binding studies on isolated membranes yielded 10-60 pmol of either 5-HT3 or P2x receptor/mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiol. responses were also detected for both SFV-5-HT3 and SFV-P2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purifn. procedure of SFV-expressed recombinant receptors, a histidine tag was introduced at the C-terminus of the 5-HT3 receptor. This 5-HT3His receptor showed high levels of expression, specific binding, and high amplitude electrophysiol. responses. For large scale expression, the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5-L fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor/mg protein corresponding to 3.2 * 106 receptors/cell. [on SciFinder (R)]

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