Lundstrom K, Michel A, Blasey H, Bernard A R, Hovius R, Vogel H, Surprenant A
Glaxo Wellcome, Medicines Research Centre, Stevenage, Herts, UK.
J Recept Signal Transduct Res. 1997 Jan-May;17(1-3):115-26. doi: 10.3109/10799899709036597.
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. Saturation binding analysis indicated the presence of more than 3 x 10(6) 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT3 or P2x receptor per mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification 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 electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 x 10(6) receptors per cell.
利用辛德毕斯病毒（SFV）表达系统表达了两种配体门控离子通道。将小鼠5-羟色胺5-HT3受体以及大鼠和人类嘌呤受体P2x亚型的cDNA导入pSFV1载体。体外转录的RNA与pSFV-Helper2 RNA共电穿孔导入BHK细胞，在体内包装后产生了高滴度的SFV-5-HT3和SFV-P2x病毒株。用这些病毒感染BHK、CHO和RIN细胞，导致重组受体的高水平表达。饱和结合分析表明每个细胞存在超过3×10⁶个5-HT3受体。对分离膜进行的结合研究显示，每毫克蛋白质含有10至60皮摩尔的5-HT3或P2x受体。在SFV感染的CHO细胞中，通过Ca²⁺动员或⁴⁵Ca²⁺内流证明了对P2x受体的功能反应。在全细胞膜片钳记录中，也检测到SFV-5-HT3和SFV-P2x感染的CHO细胞有高幅度的电生理反应。为便于纯化SFV表达的重组受体，在5-HT3受体的C末端引入了组氨酸标签。这种5-HT3His受体表现出高水平的表达、特异性结合和高幅度的电生理反应。为了大规模表达，使BHK细胞适应悬浮培养，并在11.5升发酵罐培养中用SFV-5-HT3His高效感染，从而实现高水平表达，每毫克蛋白质有52皮摩尔受体，相当于每个细胞有3.2×10⁶个受体。
