使用辛德毕斯病毒载体在哺乳动物细胞中对人α2B -肾上腺素能受体和α2B -AR-绿色荧光融合蛋白进行功能表达及直接可视化。

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors.

作者信息

Sen Saurabh, Jaakola Veli-Pekka, Heimo Heikki, Engström Mia, Larjomaa Pauliina, Scheinin Mika, Lundstrom Kenneth, Goldman Adrian

机构信息

Institute of Biotechnology (Biocenter 3), University of Helsinki, P.O. Box 65, Viikinkaari 1, FIN-00014 Helsinki, Finland.

出版信息

Protein Expr Purif. 2003 Dec;32(2):265-75. doi: 10.1016/j.pep.2003.08.006.

DOI:10.1016/j.pep.2003.08.006

PMID:14965773

Abstract

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.

摘要

α2B -肾上腺素能受体（α2B -AR）是G蛋白偶联受体（GPCR）超家族的成员之一，在哺乳动物细胞中通过辛德毕斯病毒（SFV）载体高水平表达。构建体通过将增强型绿色荧光蛋白（eGFP）和SFV衣壳融合到α2B -AR的相对末端进行工程改造。在CHO-K1细胞中表达的受体融合体α2B -AR-eGFP和CAP-α2B -AR产生的α2B值分别为176和122pmol/mg膜蛋白，并且显示出与报道的在CHO-K1细胞中稳定表达相似的配体结合特性、α2B亚型选择性和G蛋白激活。冷冻电子显微镜和基于eGFP的荧光显示了相同的亚细胞受体分布。SFV表达非常适合用于GPCR的药理学、生物化学和细胞生物学研究，以及在哺乳动物悬浮培养中大规模生产重组蛋白，以产生足够数量的受体用于结构生物学研究。