ReToAd: simple method for the rapid replacement of promoters to improve protein production
ReToAd: simple method for the rapid replacement of promoters to improve protein production
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ReToAd：快速替换启动子以提高蛋白质产量的简单方法
DOI：
10.1007/s10529-018-2541-4
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发表时间：
2018-06-01
影响因子：
2.7
通讯作者：
Zheng, Yu-Guo
中科院分区：
文献类型：
--
作者：
Cheng, Feng;Xiang, Chao;Zheng, Yu-Guo
目的建立一种快速替换启动子以提高蛋白产量的方法。结果通过用增强型绿色荧光蛋白（eGFP）替换7个IPTG诱导型启动子，建立了一种包括缺失PCR和降落PCR的快速替换启动子的方法。通过仅对30个克隆进行测序，完全回收了7个启动子。E.通过结合ReToAd和筛选实验，将携带ω-转氨酶（ω-TA）的大肠杆菌从112 U/mg细胞（T7启动子）提高到147 U/mg细胞（Trc启动子）。通过筛选含有不同启动子表达的谷氨酸脱氢酶（GDH）的文库，对E.结论构建并优化了原位快速替换启动子的“ReToAd”，一轮“ReToAd”可在3 d内完成。
Objective To develop a method for fast replacement of promoters to improve protein production.Results A method (entitled retreat to advance or "ReToAd"), which includes a deleting PCR and a touchdown PCR, was validated by replacing seven IPTG-inducible promoters with enhanced green fluorescent protein (eGFP). The seven promoters were fully recovered by sequencing only 30 clones. The activity of E. coli harboring omega-transaminase (omega-TA) was increased from 112 U/mg cells (T7 promoter) to 147 U/mg cells (Trc promoter) by combining ReToAd and screening experiments. After screening a library comprising glutamate dehydrogenase (GDH) expressed by different promoters, the activity of E. coli cell harboring Trc-promoter-expressed GDH was similar to 31-fold higher than that of T7-promoter-expressed GDH.Conclusions The "ReToAd" for in situ rapid replacement of promoters was developed and optimized, and one round of "ReToAd" can be completed within 3 days.