Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.
Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.
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DOI：
10.1073/pnas.82.23.7870
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发表时间：
1985-12
影响因子：
11.1
通讯作者：
J. D. Wet;K. Woodt;D. Helinski;Marlene DELUCAt
中科院分区：
文献类型：
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作者：
J. D. Wet;K. Woodt;D. Helinski;Marlene DELUCAt
以萤火虫（Photinus pyralis）灯笼多聚腺苷酸（poly（A）+ RNA）为模板，构建了萤火虫cDNA文库。用抗-P. pyralis荧光素酶（Photinus pyralis：oxygen 4-oxidoreductase，EC 1.13.12.7）抗体筛选文库，并分离几个表达荧光素酶抗原的cDNA克隆。一个克隆，Lambda Luc 1，含有1.5个双链酶对的cDNA，其与硝酸纤维素印迹上的1.9- 2.0-双链酶条带杂交。将克隆的cDNA与灯笼状poly（A）+ RNA杂交，选择指导体外合成单个多肽的RNA。该多肽在NaDodSO 4/PAGE上与荧光素酶共迁移，并在加入三磷酸腺苷和ATP时产生生物发光。通过用来自λ Luc 1的cDNA探测萤火虫cDNA文库来分离1.8-内切酶对cDNA。该cDNA含有足够的编码信息，指导在E.杆菌
A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.