Lundstrom K, Vargas A, Allet B
Department of Molecular Biology, Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Biochem Biophys Res Commun. 1995 Mar 8;208(1):260-6. doi: 10.1006/bbrc.1995.1332.
The 1.3 S biotinylatable subunit of Proprionibacterium shermanii transcarboxylase complex was fused to the C-terminus of the human neurokinin 1 receptor gene and introduced into the Semliki Forest virus expression vector pSFV1. RNA transcribed from pSFV1-NK1-biot and pSFV-Helper2 was coelectroporated into BHK cells permitting in vivo packaging of recombinant virus. Infection of BHK and CHO cells with SFV-NK1-biot virus yielded high level of the fusion receptor as detected by metabolic labeling, immunoblotting with streptavidin alkaline phosphatase and binding to substance P. Like native receptor, the biotinylated receptor fusion was able to stimulate Ca2+ mobilization in infected CHO cells, indicating functional coupling to guanine-nucleotide-binding proteins.
将谢氏丙酸杆菌转羧酶复合体的1.3S生物素化亚基融合到人神经激肽1受体基因的C末端，并导入辛德毕斯病毒表达载体pSFV1。从pSFV1-NK1-biot和pSFV-Helper2转录的RNA共电穿孔导入BHK细胞，以允许重组病毒在体内包装。用SFV-NK1-biot病毒感染BHK和CHO细胞，通过代谢标记、用链霉亲和素碱性磷酸酶进行免疫印迹以及与P物质结合检测到产生了高水平的融合受体。与天然受体一样，生物素化的受体融合体能够刺激被感染的CHO细胞中的Ca2+动员，表明其与鸟嘌呤核苷酸结合蛋白发生功能性偶联。
