Ray S, Zozulya S, Niemi G A, Flaherty K M, Brolley D, Dizhoor A M, McKay D B, Hurley J, Stryer L
Department of Cell Biology, Stanford University School of Medicine, CA 94305.
Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5705-9. doi: 10.1073/pnas.89.13.5705.
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.
恢复蛋白是最近发现的一种23 kDa的钙结合蛋白，当钙水平在亚微摩尔范围内降低时，它会激活视网膜视杆细胞鸟苷酸环化酶。我们在此报告从牛视网膜表达文库中克隆和测序恢复蛋白的cDNA。恢复蛋白编码序列被插入到受T7噬菌体启动子控制的pET-11a表达载体中。第二个表达系统是将编码序列置于λ噬菌体PR启动子的控制下，其产量提高了10倍（每升大肠杆菌培养物可获得10 mg纯化的恢复蛋白）。视网膜恢复蛋白在其氨基末端被肉豆蔻酰化这一发现促使我们共表达重组蛋白和N-肉豆蔻酰转移酶（EC 2.3.1.97）。在大肠杆菌中以这种方式形成的肉豆蔻酰化重组恢复蛋白与视网膜恢复蛋白相似，其色氨酸荧光发射光谱在钙诱导下发生较大变化。大量蛋白质的可获得性使我们能够将未肉豆蔻酰化的重组恢复蛋白结晶并启动X射线研究。从75%饱和度硫酸铵中获得的四方晶体的空间群为I4，晶胞尺寸a = 85.1 Å，c = 59.8 Å。这种蛋白质的钙结合形式的晶体衍射分辨率达到2.2 Å。这里描述的表达系统为对EF手超家族的这个新成员进行高分辨率X射线晶体学和核磁共振研究以及阐明其作为钙开关的精确作用模式打开了大门。
