EXPRESSION OF A SYNTHETIC BOVINE RHODOPSIN GENE IN MONKEY KIDNEY-CELLS
EXPRESSION OF A SYNTHETIC BOVINE RHODOPSIN GENE IN MONKEY KIDNEY-CELLS
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DOI：
10.1073/pnas.84.24.8874
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发表时间：
1987-12-01
影响因子：
11.1
通讯作者：
KHORANA, HG
中科院分区：
文献类型：
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作者：
OPRIAN, DD;MOLDAY, RS;KHORANA, HG
我们在这里报告的高水平表达的合成基因牛视紫红质转染猴肾COS-1细胞。视紫红质在这些细胞中产生至细胞蛋白质的0.3%的水平，并且其结合外源添加的11-顺式-视黄醛以产生特征性视紫红质吸收光谱。我们描述了一个一步的免疫亲和程序，用于纯化的视紫红质基本上是同质的。COS-1细胞视紫红质以光依赖的方式激活牛转导素的GT3活性，其比活性与纯化的牛视紫红质相同。免疫金染色细胞的电子显微镜检查表明，视紫红质位于转染细胞的质膜上，并与膜的细胞外侧上的氨基末端定向。这种取向类似于牛视网膜感光细胞盘膜中的视紫红质。
We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cells rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.