Shigesada K, Tsurushita N, Matsumoto Y, Imai M
Gene. 1984 Jul-Aug;29(1-2):199-209. doi: 10.1016/0378-1119(84)90180-x.
A plasmid system has been constructed which allows high-level expression of the rho gene of Escherichia coli under the control of the pL promoter and the N-antitermination regulatory system of bacteriophage lambda. The pL-directed synthesis of Rho crucially depends on the lambda N gene product and is promoted most effectively when this product is supplied from the N gene cloned on a separate compatible plasmid with a moderate copy number. The requirement for N can be circumvented partly, but not completely, by deletion of the region preceding the rho structural gene. Attempts were also made to optimize the construction of rho-expression plasmids by adjusting the orientation and location of pL and rho inserts on the pBR322 vector. With optimal conditions, Rho protein is overexpressed 100-fold and can become as much as 10% of the total cellular protein. Using this plasmid system, Rho can be purified with a yield of more than 20 mg from 10 g of induced cells.
构建了一种质粒系统，该系统可在噬菌体λ的pL启动子和N-抗终止调节系统的控制下，实现大肠杆菌rho基因的高水平表达。由pL指导的Rho合成关键取决于λN基因产物，当该产物由克隆在具有中等拷贝数的单独相容质粒上的N基因提供时，其合成最为有效。通过缺失rho结构基因之前的区域，可以部分但不能完全规避对N的需求。还尝试通过调整pL和rho插入片段在pBR322载体上的方向和位置来优化rho表达质粒的构建。在最佳条件下，Rho蛋白的表达量可超过100倍，可占细胞总蛋白的10%。使用该质粒系统，从10 g诱导细胞中可纯化出产量超过20 mg的Rho。
