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) atvarious 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
