Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.
Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.
复制标题
视觉钙传感器恢复蛋白的克隆、表达和结晶。
DOI：
10.1073/pnas.89.13.5705
复制
发表时间：
1992
影响因子：
11.1
通讯作者：
Stryer,L
中科院分区：
文献类型：
--
作者：
Ray,S;Zozulya,S;Niemi,GA;Flaherty,KM;Brolley,D;Dizhoor,AM;McKay,DB;Hurley,J;Stryer,L
恢复蛋白，最近发现的23 kDa的钙结合蛋白，激活视网膜杆鸟苷酸环化酶时，钙水平降低在亚微摩尔范围。本文报道了从牛视网膜表达文库中克隆的一个recoverin基因的cDNA序列。将回收素编码序列插入到T7噬菌体启动子控制下的pET-11 a表达载体中。第二个表达系统，其中编码序列被置于λ噬菌体PR启动子的控制下，给出了10倍更高的产量（每升大肠杆菌培养物10 mg纯化的回收素）。视网膜恢复蛋白在其氨基末端被肉豆蔻酰化的发现使我们共表达重组蛋白和N-肉豆蔻酰转移酶（EC 2.3.1.97）。在E.大肠杆菌的色氨酸荧光发射光谱与视网膜恢复蛋白相似，表现出钙诱导的大位移。丰富的蛋白质的可用性使我们能够结晶未豆蔻酰化的重组回收蛋白，并开始X射线研究。从75%饱和硫酸铵溶液中得到的四氢呋喃晶体空间群为I4，晶胞尺寸a = 85.1 A，c = 59.8 A。这些钙结合形式的蛋白质晶体衍射到2.2 A的分辨率。这里描述的表达系统打开了大门，高分辨率的X-射线晶体学和核磁共振研究的EF-手超家族的这个新成员，并阐明其精确的作用模式作为钙开关。
Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.
影响因子：
4.3
作者：
H. Sambrook
通讯作者：
H. Sambrook