Fürste J P, Pansegrau W, Frank R, Blöcker H, Scholz P, Bagdasarian M, Lanka E
Gene. 1986;48(1):119-31. doi: 10.1016/0378-1119(86)90358-6.
Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species. Analysis of the products in E. coli revealed that in addition to the two primase polypeptides of 118 and 80 kDa the pri region of RP4 encodes two smaller proteins of 16.5 and 8.6 kDa. The transcript for the four RP4-specified products is polycistronic. The vector system used in E. coli is based on the plasmid pKK223-3 (Brosius and Holy, 1984), a ColE1-type replicon which contains a polylinker sequence flanked on one side by the controllable tac promoter and on the other side by two strong transcriptional terminators. The gene for the lac repressor (lacIQ) was inserted to render the use of the plasmid independent from repressor-overproducing strains. The gene cartridge essential for high-level expression and selection was combined with the RSF1010 replicon to generate a vector plasmid functioning in a wide variety of Gram-negative hosts. The versatility of the vector family was extended by constructing derivatives that contain the polylinker in inverted orientation relative to the tac promoter. Therefore, the orientation of the cloned fragment can be chosen by 'forced cloning' into the appropriately selected vector.
利用自调控的高水平表达载体系统，在大肠杆菌及其他四种革兰氏阴性细菌中过量表达了质粒RP4引发酶。对大肠杆菌中的产物分析表明，除了118 kDa和80 kDa的两种引发酶多肽外，RP4的pri区域还编码16.5 kDa和8.6 kDa的两种较小蛋白质。RP4指定的这四种产物的转录本是多顺反子的。在大肠杆菌中使用的载体系统基于质粒pKK223 - 3（Brosius和Holy，1984），这是一种ColE1型复制子，其包含一个多克隆位点序列，一侧由可控的tac启动子侧翼，另一侧由两个强转录终止子侧翼。插入了乳糖阻遏物基因（lacIQ），以使质粒的使用不依赖于阻遏物过量表达的菌株。将高水平表达和选择所必需的基因盒与RSF1010复制子组合，以产生在多种革兰氏阴性宿主中起作用的载体质粒。通过构建相对于tac启动子以反向方式包含多克隆位点的衍生物，扩展了载体家族的通用性。因此，可以通过“强制克隆”到适当选择的载体中来选择克隆片段的方向。
