Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.
Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.
复制标题
最大化含有 lambda PL 启动子的质粒载体的基因表达：过量产生转录终止因子 rho 的策略。
DOI：
10.1073/pnas.82.1.88
复制
发表时间：
1985
影响因子：
11.1
通讯作者：
Platt,T
中科院分区：
文献类型：
--
作者：
Mott,JE;Grant,RA;Ho,YS;Platt,T
我们已经构建了两个质粒，其中来自大肠杆菌K-12的rho基因的转录在λ噬菌体PL启动子的控制下。在p31-356中，正常的rho启动子被删除，但剩余的rho前导区，包括核糖体结合位点，是存在的。在p39-AS中，rho前导序列完全缺失，λ cII核糖体结合位点取代rho。在非诱导条件下，这些质粒中rho蛋白的表达被携带λ隐蔽前噬菌体的宿主中的λ cI蛋白抑制。在携带cI+阻遏物的隐蔽溶原细胞中使用丝裂霉素C或萘啶酸进行诱导，导致rho蛋白的过量产生，p31-356的rho蛋白水平达到总细胞蛋白的3%-5%，p39-AS的rho蛋白水平约等于40%。过量产生的蛋白质在功能上与从K-12菌株W3110中分离的rho蛋白没有区别，并且可以从携带p39-AS的细胞中获得，产量高达25 mg rho/g细胞。与化学诱导相反，在四个隐蔽的λ溶原菌携带热不稳定的cI 857阻遏物的热诱导未能产生相同的高水平的rho蛋白（与任何质粒）。我们的研究结果表明，化学诱导含PL的质粒表达载体可以作为一个方便和有用的替代常用的方法热诱导。
We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present. In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho. Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS. The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.