Bernard H U, Remaut E, Hershfield M V, Das H K, Helinski D R, Yanofsky C, Franklin N
Gene. 1979 Jan;5(1):59-76. doi: 10.1016/0378-1119(79)90092-1.
Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.
构建了两种多拷贝的、ColE1 型质粒克隆载体 pHUB2 和 pHUB4，它们在噬菌体 λ 启动子 pL 的下游带有四个不同的单限制酶切位点。在存在指定温度敏感阻遏物的 cIts 基因时，pL 的促进活性在低温下被关闭，但可通过热诱导激活。cIts 位于宿主染色体上，或位于与克隆载体相容的第二个质粒 pRK248 上，或位于载体本身。分别将三个不同的限制酶切片段插入这些质粒的 EcoRI、BamHI 和 SalI 位点，每个片段携带鼠伤寒沙门氏菌或痢疾志贺氏菌的 trpA 基因，并通过测量 trpA 基因产物的酶活性来确定插入基因在大肠杆菌中 pL 依赖性表达。热诱导导致 trpA 的表达水平相当于总可溶性细胞蛋白的 1%至 6.6%，以 trpA 蛋白形式存在。trpA 蛋白的产生水平取决于特定的插入片段和所使用的质粒。
