CONSTRUCTION OF PLASMID CLONING VEHICLES THAT PROMOTE GENE-EXPRESSION FROM THE BACTERIOPHAGE LAMBDA-PL PROMOTER
CONSTRUCTION OF PLASMID CLONING VEHICLES THAT PROMOTE GENE-EXPRESSION FROM THE BACTERIOPHAGE LAMBDA-PL PROMOTER
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DOI：
10.1016/0378-1119(79)90092-1
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发表时间：
1979-01-01
期刊：
影响因子：
3.5
通讯作者：
HELINSKI, DR
中科院分区：
文献类型：
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作者：
BERNARD, HU;REMAUT, E;HELINSKI, DR
构建了两个多拷贝ColE 1型质粒克隆载体pHUB 2和pHUB 4，它们在噬菌体的下游携带4个不同的单限制性位点。启动子pL. pL的促进活性在低温下在cIts基因的存在下被关闭，所述cIts基因指定温度敏感性阻遏物，但可以通过热诱导被激活。cIt位于宿主染色体上，或与克隆载体相容的第二质粒pRK 248上，或载体本身上。将三种不同的限制性片段分别插入到这些质粒的EcoRI、BamHI和SalI位点中，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的pL依赖性表达，所述三种不同的限制性片段各自携带鼠伤寒沙门氏菌或志贺氏菌的基因trpA。热诱导导致trpA的表达水平相当于总可溶性细胞蛋白的1-6.6%作为trpA蛋白。trpA蛋白的产生水平取决于特定的插入片段和所用的质粒。
Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, were constructed that carry 4 different single restriction sites downstream 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 2nd 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, were 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 1-6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.