Brünen-Nieweler C, Meyer F, Heckmann K
Institut für Allgemeine Zoologie und Genetik, Universität Münster, Germany.
Gene. 1994 Dec 2;150(1):187-92. doi: 10.1016/0378-1119(94)90882-6.
The pheromone 3-encoding gene (phr3) of Euplotes octocarinatus was expressed in Escherichia coli using a novel expression-secretion vector. The vector, pExSec1, contains a strong and tightly regulated T7 promoter, the corresponding Shine-Dalgarno sequence and the T7 terminator region. Translation starts at the protein A leader sequence followed by the synthetic ZZ sequence of protein A. The expression-secretion modules are embedded in the high-copy-number plasmid vector, pUK21, which carries a kanamycin-resistance marker (KmR). The produced ZZ-pheromone 3 (Phr3) fusion protein was secreted into the culture medium of the host cells. It was isolated by affinity chromatography and was further purified by gel filtration. After refolding with protein disulfide isomerase (PDI), the fusion protein exhibited the same high activity as the native pheromone.
利用一种新型表达分泌载体，在大肠杆菌中表达了八肋游仆虫的信息素3编码基因（phr3）。该载体pExSec1包含一个强且调控严格的T7启动子、相应的SD序列和T7终止子区域。翻译起始于蛋白A前导序列，其后是蛋白A的合成ZZ序列。表达分泌模块嵌入高拷贝数质粒载体pUK21中，该载体携带卡那霉素抗性标记（KmR）。所产生的ZZ-信息素3（Phr3）融合蛋白分泌到宿主细胞的培养基中。通过亲和层析进行分离，并通过凝胶过滤进一步纯化。用蛋白二硫键异构酶（PDI）复性后，融合蛋白表现出与天然信息素相同的高活性。
