Buckel P, Zehelein E
Gene. 1981 Dec;16(1-3):149-59. doi: 10.1016/0378-1119(81)90071-8.
To investigate the heterologous expression of Pseudomonas genes in Escherichia coli we have cloned P. fluorescens DNA in an E. coli [cosmid] system. A colony bank representing the whole P. fluorescens chromosome was screened immunologically using a modification of the method described by Broome and Gilbert (1978). Radioactive labelling of the antibodies was replaced by conjugation with horseradish peroxidase. Among 523 E. coli colonies one was D-galactose dehydrogenase-positive. The expression of this enzyme in primary clones was lower than in the uninduced Pseudomonas. Subcloning of the D-galactose dehydrogenase gene, in vitro mutagenesis of the DNA, and coupling to a strong E. coli promoter yielded an E. coli strain that produces 90 times more of the enzyme than the induced P. fluorescens.
为了研究假单胞菌基因在大肠杆菌中的异源表达，我们已将荧光假单胞菌的DNA克隆到大肠杆菌[黏粒]系统中。使用对Broome和Gilbert（1978年）所述方法的一种改进，通过免疫筛选代表整个荧光假单胞菌染色体的菌落文库。用辣根过氧化物酶偶联取代抗体的放射性标记。在523个大肠杆菌菌落中，有一个菌落的D-半乳糖脱氢酶呈阳性。该酶在初级克隆中的表达低于未诱导的假单胞菌。D-半乳糖脱氢酶基因的亚克隆、DNA的体外诱变以及与强大肠杆菌启动子的偶联产生了一种大肠杆菌菌株，该菌株产生的这种酶比诱导的荧光假单胞菌多90倍。
