Induction of T7 Promoter at Higher Temperatures May Be Counterproductive
Bacterial expression of recombinant proteins is the most popular and convenient method for obtaining large quantities of pure protein. The induction of T7 promoter with isopropyl-β-d-thiogalactopyranoside (IPTG) is widely used for expression of large quantities of proteins in Escherichia coli. It has been reported that basic T7 promoter is leaky and expresses cloned genes without induction. The effect of T7 promoter induction on expression of proteins at different temperature using flow cytometry has not yet been investigated. Green fluorescent protein (GFP) as a non-peptide tag can be used for protein solubility screening and for high-throughput optimization of expression conditions using flow cytometry. Therefore, flow cytometry was used to study the effect of induction on the expression of T7 promoter driven emerald GFP (emGFP) at various temperatures. We noticed that percentage of emGFP positive cells decreased instead of increasing upon induction at higher temperatures. Western blot analysis confirmed that the amount of total and soluble emGFP decreased in induced cells compared uninduced cells at higher temperatures. Our results indicate that induction of basic T7 promoter at higher temperature may not necessarily increase protein expression. While using a basic T7 promoter it is highly recommended to analyze the effect of induction on protein expression at various temperatures.
KeywordsExpression Flow cytometry Induction T7 promoter emGFP
This work was financially supported by UGC-BSR research startup Grant (F, 30-12/2014 (BSR)), Govt. of India, sanctioned to RA. We acknowledge Sophisticated Instrument Centre (SIC), Dr. Harisingh Gour Central University, Sagar (MP)-India for providing Flow Cytometer facility. We also would like to thank Dr. Shivendra K. Chourasia, Department of Microbiology, Dr. Harisingh Gour Central University, Sagar (MP)-India for providing invaluable inputs and instrumental facility. PN thanks UGC-CSIR for research fellowship.
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Conflict of interest
All the author have contributed to the manuscript and consented for the submission and declare no conflict of interest.
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