Pradel E, Boquet P L
Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur Yvette, France.
J Bacteriol. 1988 Oct;170(10):4916-23. doi: 10.1128/jb.170.10.4916-4923.1988.
Several unknown Escherichia coli genes for different species of acid phosphatase were cloned in vivo with the plasmid Mu dII4042. When present in a multicopy state, each gene promoted hydrolysis of p-nitrophenyl-phosphate at acidic pH. Among seven recombinant clones that encoded periplasmic acid phosphatase activities, five different genes could be distinguished by the pH optimum and substrate preference for the enzyme and by the restriction enzyme pattern. A 1.7-kilobase recombinant DNA fragment, common to two clones, was inserted into plasmid pBR322 and shown to contain a new gene, agp, which leads to the overexpression of the periplasmic acid glucose-1-phosphatase, a dimer of a 44-kilodalton polypeptide. Fusions of agp to gene phoA deprived of its own signal sequence conferred an alkaline phosphatase-positive phenotype to bacteria, showing the presence of an export signal on agp. The resulting hybrid proteins were characterized by immunoprecipitation with an antiserum directed against purified acid phosphatase or against alkaline phosphatase, showing that agp is the structural gene of the acid phosphatase. The beginning, the orientation, and the end of gene agp on the cloned DNA fragment were determined by the characteristics of such hybrid proteins.
利用质粒Mu dII4042在体内克隆了几种不同物种的未知大肠杆菌酸性磷酸酶基因。当以多拷贝状态存在时，每个基因都能在酸性pH条件下促进对硝基苯磷酸酯的水解。在七个编码周质酸性磷酸酶活性的重组克隆中，根据酶的最适pH值、底物偏好以及限制性内切酶图谱，可以区分出五个不同的基因。将两个克隆共有的一个1.7千碱基的重组DNA片段插入质粒pBR322，结果表明该片段含有一个新基因agp，它能导致周质酸性葡萄糖-1-磷酸酶的过表达，该酶是一种由44千道尔顿多肽组成的二聚体。将agp与缺失自身信号序列的phoA基因融合，使细菌呈现碱性磷酸酶阳性表型，表明agp上存在输出信号。通过用针对纯化的酸性磷酸酶或碱性磷酸酶的抗血清进行免疫沉淀来表征所得的杂合蛋白，结果表明agp是酸性磷酸酶的结构基因。根据此类杂合蛋白的特性确定了克隆DNA片段上agp基因的起始、方向和末端。
