中华医学杂志（英文版）2002，Vol.115Issue(12)：1785-1789,5.
操纵子重复克隆以提高外源基因的表达水平及同一大肠杆菌细胞内质粒DNA总量为一常数的新概念
High-level expression of foreign genes via multiple joined operons and a new concept regarding the restricted constant of total amount of plasmid DNA per Escherichia coli cell
摘要
Abstract
Objective To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell.Methods Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with 3 H-thymidine (3H-TdR). Results No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9%±3.9%, 51.3%±4.1%, 54.8%±3.3% and 58.2%±3.4% of total cell protein. In the CW12 series, the yields were 32.2%±5.0%, 42.8%±4.1% and 46.9%±4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P<0.01). Further calculation showed that the total amount of plasmid DNA per cell was not significantly different in each series (P>0.05) and restricted to some extent. Conclusions Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.关键词
操纵子/基因剂量/基因表达/质粒拷贝数/限制性常数Key words
operon/gene dosage/gene expression/plasmid copy number/restricted constant分类
医药卫生引用本文复制引用
陈伟京,洪梅,李丹,卢圣栋..操纵子重复克隆以提高外源基因的表达水平及同一大肠杆菌细胞内质粒DNA总量为一常数的新概念[J].中华医学杂志（英文版）,2002,115(12):1785-1789,5.基金项目
This study was supported by a grant from the National High Technology Research and Development Program (No. 102-08-02-02). （No. 102-08-02-02）
