Induction of T7 Promoter at Higher Temperatures May Be Counterproductive

Indian Journal of Clinical Biochemistry : IJCB
Priyanka NamdevRajaneesh Anupam

Abstract

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 increas...Continue Reading

References

Apr 1, 1968·Proceedings of the National Academy of Sciences of the United States of America·B Müller-HillW Gilbert
Jul 15, 1999·Nature Biotechnology·G S WaldoT C Terwilliger
Nov 22, 2005·Nature Methods·Nathan C ShanerRoger Y Tsien
Dec 22, 2005·Nature Biotechnology·Jean-Denis PédelacqGeoffrey S Waldo
Jan 29, 2013·International Review of Cell and Molecular Biology·Olesya V StepanenkoKonstantin K Turoverov

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Methods Mentioned

BETA
flow cytometry
Fluorescence
electrophoresis

Software Mentioned

AlphaEase FC 2200
[UNK]
Flowjo

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