ReToAd: simple method for the rapid replacement of promoters to improve protein production
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To develop a method for fast replacement of promoters to improve protein production.
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.
The “ReToAd” for in situ rapid replacement of promoters was developed and optimized, and one round of “ReToAd” can be completed within 3 days.
KeywordsIPTG-inducible promoters Protein production Replacing promoters ReToAd
This work was financially supported by Natural Science Foundation of China (31700693), Zhejiang Provincial Natural Science Foundation of China (LQ17C050002) and China Postdoctoral Science Foundation (2017M612030).
Supplementary Table 1—Oligonucleotide sequences used for in removing and replacing promoters.
Supplementary Table 2—Oligonucleotide sequences used for sequencing.
Supplementary Figure 1—Maps of starting plasmids.
Supplementary Figure 2—ω-TA expression levels by different promoters.
Additional method 1—Expression levels of eGFP, ω-TA, NIT and GDH in MTPs or flasks.
Additional method 2—Protein analysis.
Additional method 3—Activity analysis of ω-transaminase, nitrilase, and glutamate dehydrogenase.
Compliance with ethical standards
Conflict of interest
The authors declare no financial or commercial conflict of interest.
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