EXPRESSION OF A CLONED STREPTAVIDIN GENE IN ESCHERICHIA-COLI
EXPRESSION OF A CLONED STREPTAVIDIN GENE IN ESCHERICHIA-COLI
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DOI：
10.1073/pnas.87.1.142
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发表时间：
1990-01-01
影响因子：
11.1
通讯作者：
CANTOR, CR
中科院分区：
文献类型：
--
作者：
SANO, T;CANTOR, CR
我们描述了用于在大肠杆菌中表达克隆的链霉亲和素基因的系统的构建。尽管链霉亲和素基因对宿主细胞具有极强的致死性，但由于基因产物与生物素的强结合，该基因在大肠杆菌中得到了高效表达。大肠杆菌中表达。表达的链霉亲和素占菌体总蛋白的35%以上。表达的链霉亲和素不溶于细胞。然而，通过对6 M盐酸胍（pH 1.5）透析溶解并通过透析除去盐酸胍后，蛋白质变得可溶并复性。这个简单的过程产生纯化的链霉亲和素几乎同质。纯化的链霉亲和素结合3.5-3.9个生物素分子/分子，表明其具有几乎完全的生物素结合能力。一些纯化的链霉亲和素分子聚集成低聚物，这表明存在于我们的材料中但在典型制剂中不存在的分子的C-末端区域可能是导致聚集的原因。
We describe the construction of systems for expressing the cloned streptavidin gene in Escherichia coli. Although the streptavidin gene is extremely lethal to the host cells, because of the strong biotin binding of the gene product, the gene was expressed efficiently in E. coli by using T7 RNA polymerase/T7 promoter expression systems. The expressed streptavidin accumulated to more than 35% of the total cell protein. The expressed streptavidin was insoluble in the cell. However, after solubilization by dialysis against 6 M guanidine hydrochloride (pH 1.5) and removal of guanidine hydrochloride by dialysis, the protein became soluble and renatured. This simple procedure yielded streptavidin purified almost to homogeneity. The purified streptavidin bound 3.5-3.9 molecules of biotin per molecule, indicating that it had almost full biotin-binding ability. Some of the purified streptavidin molecules aggregated into oligomers, suggesting that the C-terminal region of the molecule, present in our material but absent in typical preparations, may be responsible for the aggregation.