Feng Cheng1, Chao Xiang1, Xiao-Jian Zhang1, Zhi-Qiang Liu2, Yu-Guo Zheng1. 1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People's Republic of China. 2. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People's Republic of China. microliu@zjut.edu.cn.
Abstract
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 ω-transaminase (ω-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 ~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.
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 ω-transaminase (ω-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 ~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.
Entities:
Keywords:
IPTG-inducible promoters; Protein production; ReToAd; Replacing promoters