Inouye S, Zenno S, Sakaki Y, Tsuji F I
Yokohama Research Center, Chisso Corporation, Japan.
Protein Expr Purif. 1991 Apr-Jun;2(2-3):122-6. doi: 10.1016/1046-5928(91)90060-v.
A fairly rapid and improved method for producing large amounts of highly pure apoaequorin, the apoprotein of aequorin which emits light on binding Ca2+, is described. The method consists of fusing the gene of the outer membrane protein A (ompA) secretion signal peptide of Escherichia coli to the apoaequorin gene and expressing the fused gene in the bacterium. The expressed protein is correctly cleaved in the process of being secreted across the cell membrane into the culture medium. The apoaequorin is subsequently purified by acid precipitation and DEAE-cellulose chromatography, yielding a product of greater than 95% purity. The availability of pure apoaequorin makes possible detailed studies of the physical-chemical properties of this Ca2(+)-binding protein and allows for the preparation of pure aequorin for use in highly specific and sensitive assays for Ca2+.
本文描述了一种相当快速且改良的方法，用于大量生产高度纯化的脱辅基水母发光蛋白，脱辅基水母发光蛋白是水母发光蛋白的脱辅基蛋白，在结合Ca2+时会发光。该方法包括将大肠杆菌外膜蛋白A（ompA）分泌信号肽的基因与脱辅基水母发光蛋白基因融合，并在细菌中表达融合基因。表达的蛋白质在跨细胞膜分泌到培养基的过程中被正确切割。随后通过酸沉淀和DEAE-纤维素色谱法纯化脱辅基水母发光蛋白，得到纯度大于95%的产物。纯脱辅基水母发光蛋白的可得性使得对这种Ca2+结合蛋白的物理化学性质进行详细研究成为可能，并允许制备用于Ca2+高度特异性和灵敏测定的纯水母发光蛋白。
