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

摘要

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.%目的确定在一个质粒载体上串联目的操纵子以提高目的蛋白表达量的可行性,阐明宿主细胞对胞内质粒DNA总量调控的可能机制.方法亚克隆构建了两组操纵子正向串联的表达质粒:CWll系列分别含1-4个正向操纵子,质粒大小以2.25 kb的增加量从5.47 kb增加至12.26 kb;CW12系列分别含1-3个正向操纵子,质粒大小以2.16 kb的增加量从5.40 kb增加至9.72 kb.SDS凝胶电泳和激光密度扫描测定目的蛋白表达量;3H-TdR掺入法测定质粒拷贝数.结果操纵子的串联不影响宿主大肠杆菌的生长;温度诱导表达后CW11系列目的蛋白表达量分别为菌体总蛋白的44.9%±3.9%、51.3%±4.1%、54.8%±3.3%和58.2%±3.4%,CW12系列目的蛋白表达量分别为菌体总蛋白的32.2%±5.0%、42.8%±4.1%和46.9%±4.0%.两组质粒的拷贝数均随操纵子串联个数的增加而显著减少(P<0.01),但目的基因的总剂量随之显著增加(P<0.01),而同一系列的质粒在每个宿主细胞内的质粒DNA总量没有显著的变化(P>0.05).结论操纵子串联增加了目的基因的剂量从而提高了目的基因在大肠杆菌中的表达水平.质粒大小和其拷贝数呈负相关,在同一培养条件下,对于特定的大肠杆菌菌株,同一系列的质粒在宿主细胞内的DNA总量在一定程度上相对恒定.