Togna A P, Shuler M L, Wilson D B
School of Chemical Engineering, Cornell University, Ithaca, New York 14853.
Biotechnol Prog. 1993 Jan-Feb;9(1):31-9. doi: 10.1021/bp00019a005.
Runaway plasmid replication can be used to increase target gene dosage and thereby overproduce proteins within the bacterium Escherichia coli. However, the presence of excessive plasmid DNA often alters normal cell functions. High copy number plasmids with strong promoters place a severe metabolic burden on the cell, causing a decreased specific growth rate and changes in cell physiology. Induction of beta-lactamase synthesis from the tac promoter on plasmid pKN causes runaway plasmid replication and excretion of beta-lactamase. Runaway plasmid replication results from readthrough of tac promoter transcripts into the replication region of the plasmid. Both high plasmid copy numbers and a strong promoter (tac) are necessary to achieve the level of overproduction necessary for excretion of beta-lactamase, but high-level target protein synthesis is detrimental to the cell. A derivative of pKN which is more easily regulated was constructed by adding the lacI gene to the plasmid.
失控质粒复制可用于增加靶基因剂量，从而在大肠杆菌中过量生产蛋白质。然而，过量质粒DNA的存在常常会改变正常细胞功能。带有强启动子的高拷贝数质粒会给细胞带来严重的代谢负担，导致比生长速率降低和细胞生理变化。从质粒pKN上的tac启动子诱导β-内酰胺酶合成会导致质粒失控复制和β-内酰胺酶的分泌。失控质粒复制是由于tac启动子转录本通读进入质粒的复制区域所致。高质粒拷贝数和强启动子（tac）都是实现β-内酰胺酶分泌所需的过量生产水平所必需的，但高水平的靶蛋白合成对细胞是有害的。通过向质粒中添加lacI基因构建了一种更易于调控的pKN衍生物。
