Diederich L, Roth A, Messer W
Max-Planck-Institut für molekulare Genetik, Berlin, FRG.
Biotechniques. 1994 May;16(5):916-23.
A series of plasmid expression vectors, which support the regulated and efficient expression of genes in Escherichia coli, have been constructed. The vectors consist of a DNA replication origin cassette, a promoter cassette, an efficient ribosome binding site together with a polylinker region and a lacZ gene. Several types of replication origins and promoter sequences are each available on cassettes. Fusion of the 5' TG dinucleotide of the gene under consideration to the A nucleotide, present on the vector, results in an ATG start codon and allows, in combination with the plasmid-borne ribosome binding site, the efficient expression of the cloned gene. Additionally, a second fusion of the gene at its 3' end with the lacZ gene, which is available in all three reading frames relative to the polylinker region, allows rapid selection of the correctly fused genes. As an example of the cloning of a regulatory gene, this vector system was used for the expression of the dnaA gene, of Escherichia coli, the initiator protein for DNA replication.
已构建了一系列质粒表达载体，可支持基因在大肠杆菌中进行调控且高效的表达。这些载体由一个DNA复制起点盒、一个启动子盒、一个高效核糖体结合位点以及一个多克隆位点区域和一个lacZ基因组成。几种类型的复制起点和启动子序列在盒中均有提供。将所研究基因的5'端TG二核苷酸与载体上的A核苷酸融合，可形成一个ATG起始密码子，并与质粒携带的核糖体结合位点相结合，从而实现克隆基因的高效表达。此外，基因在其3'端与lacZ基因的第二次融合，相对于多克隆位点区域，该融合在所有三个阅读框中均可实现，这使得能够快速筛选出正确融合的基因。作为调控基因克隆的一个例子，该载体系统被用于表达大肠杆菌的dnaA基因，即DNA复制的起始蛋白。
