Interplay of SOS induction, recombinant gene expression, and multimerization of plasmid vectors in Escherichia coli
Interplay of SOS induction, recombinant gene expression, and multimerization of plasmid vectors in Escherichia coli
复制标题
DOI：
10.1002/bit.10354
复制
发表时间：
2002-10-05
影响因子：
3.8
通讯作者：
Park, YH
中科院分区：
文献类型：
--
作者：
Lee, J;Kim, HC;Park, YH
使用 pBR322 和 pUC 衍生的质粒载体，在大肠杆菌 IC2015(recA::lacZ) 和 GY4786 (sfiA::lacZ) 菌株中合成同源蛋白（大肠杆菌天然酯酶）和三种异源蛋白（人白细胞介素 2、人白细胞介素 6 和发酵单胞菌果聚糖蔗糖酶）。通过对每种重组培养物中 β-半乳糖苷酶活性的时间过程测量，详细评估了 SOS 诱导，并对结果进行了系统比较。在重组大肠杆菌中，无论是单独的重组插入阴性质粒主链还是含有同源基因的表达载体都不会发生SOS反应。无论合成的外源蛋白的基因表达水平和毒性活性如何，只有当使用特定的质粒载体表达异源基因时才会诱导SOS反应，这表明对重组基因克隆和质粒载体系统的选择有很强的依赖性。有人认为，在重组大肠杆菌中，SOS 反应（即，recA 表达和初始 sfiA 表达的激活）可能与合成的异源蛋白的代谢负担或毒性细胞事件无关，但可能是由外源基因与质粒载体之间的外源基因特异性相互作用引起的。与所使用的大肠杆菌XL1-blue(recA(-))菌株不同，编码三种异源蛋白中每一种的所有表达载体在培养过程中在大肠杆菌IC2015菌株中发生多聚化，而含有同源基因的表达载体从未形成质粒多聚体。多聚化的程度还取决于表达载体中的外源基因插入。作为 SOS 诱导的显着效应，用于异源蛋白表达的重组质粒载体似乎在 recA+ 大肠杆菌宿主中显着形成各种多聚体。 (C) 2002 年 Wiley 期刊公司
Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA+ E. coli host. (C) 2002 Wiley Periodicals, Inc.