Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli
Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli
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开发用于在大肠杆菌中克隆和表达剧毒基因的新型载体
DOI：
10.1016/j.ejbt.2017.10.004
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发表时间：
2017-11-15
影响因子：
2.7
通讯作者：
Xu, Daqing
中科院分区：
文献类型：
--
作者：
Li, Hedan;Hao, Chengwei;Xu, Daqing
背景：大肠杆菌已被广泛用作克隆和表达异源基因的宿主。然而，目前可用于克隆和表达极毒基因的载体很少，限制了对极毒蛋白的进一步基础和应用研究。杆菌pAU 10利用有效但高度可抑制的T7-lacO启动子/操纵子和T7启动子上游的强rrnBT 2转录终止子的组合，以严格控制极毒基因的不需要的转录;此外，trp启动子/操纵子与T7启动子相对定向，以控制可阻断渗漏mRNA翻译的反义RNA的产生。在培养基中不添加IPTG和L-色氨酸的情况下，T7启动子对极毒基因的转录被高度抑制，并且trp启动子产生反义RNA，这严格阻止了极毒蛋白在E.杆菌在添加IPTG和L-色氨酸的条件下，T7启动子能够高效转录该基因，而trp启动子不产生反义RNA，从而保证了该基因在大肠杆菌中的高效表达。杆菌通过在大肠杆菌中克隆和表达限制性内切酶编码基因bamHI而不克隆相应的甲基化酶基因，证实了克隆到载体pAU 10中的一个极毒基因的紧密调控和表达效率。结论：pAU 10是一个良好的表达载体，可用于大肠杆菌JM 109（DE 3）中的极毒基因的克隆和表达。杆菌(C)2017年瓦尔帕莱索天主教大学制作和主办Elsevier B. V.保留所有权利。
Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins.Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3).Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli. (C) 2017 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved.