USE OF TNPHOA TO DETECT GENES FOR EXPORTED PROTEINS IN ESCHERICHIA-COLI - IDENTIFICATION OF THE PLASMID-ENCODED GENE FOR A PERIPLASMIC ACID-PHOSPHATASE
USE OF TNPHOA TO DETECT GENES FOR EXPORTED PROTEINS IN ESCHERICHIA-COLI - IDENTIFICATION OF THE PLASMID-ENCODED GENE FOR A PERIPLASMIC ACID-PHOSPHATASE
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DOI：
10.1128/jb.169.4.1663-1669.1987
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发表时间：
1987-04-01
影响因子：
3.2
通讯作者：
BECKWITH, J
中科院分区：
文献类型：
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作者：
BOQUET, PL;MANOIL, C;BECKWITH, J
采用体内和体外相结合的技术，将大肠杆菌周质酸性磷酸酶（最适2.5）的结构基因（appA）克隆到质粒中。通过使用与由Tn5 IS50 L：：phoA（TnphoA）插入产生的碱性磷酸酶基因（phoA）的融合物来鉴定appA基因在克隆的DNA片段内的位置和方向。为了使TnphoA产生的杂合蛋白具有高酶活性，似乎phoA基因必须与编码促进蛋白输出的信号的靶基因融合。因此，用于鉴定appA基因的方法似乎提供了选择性地鉴定编码膜蛋白和分泌蛋白的基因的简单的一般手段。
The structural gene (appA) for the periplasmic acid phosphatase(optimum 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques. The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions. For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export. The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.