Boquet P L, Manoil C, Beckwith J
J Bacteriol. 1987 Apr;169(4):1663-9. doi: 10.1128/jb.169.4.1663-1669.1987.
The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 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.
利用体内和体外技术相结合的方法，将大肠杆菌周质酸性磷酸酶（最适pH 2.5）的结构基因（appA）克隆到一个质粒中。通过使用由Tn5 IS50L::phoA（TnphoA）插入产生的与碱性磷酸酶基因（phoA）的融合体，确定了克隆DNA片段内appA基因的位置和方向。对于由TnphoA产生的杂合蛋白要有高酶活性，phoA基因似乎必须与编码促进蛋白质输出信号的靶基因融合。因此，用于鉴定appA基因的方法似乎提供了一种简单通用的手段，可选择性地鉴定编码膜蛋白和分泌蛋白的基因。
