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FEMS Microbiol Lett. 2009 Oct;299(1):86-94. doi: 10.1111/j.1574-6968.2009.01738.x. Epub 2009 Jul 27.

Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins.

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  • 1School of Biosciences, University of Birmingham, Birmingham, UK School of Chemical Engineering, University of Birmingham, Birmingham, UK.

Abstract

A C-terminal green fluorescent protein (GFP) fusion to a model target protein, Escherichia coli CheY, was exploited both as a reporter of the accumulation of soluble recombinant protein, and to develop a generic approach to optimize protein yields. The rapid accumulation of CheY∷GFP expressed from a pET20 vector under the control of an isopropyl-β-d-thiogalactoside (IPTG)-inducible T7 RNA polymerase resulted not only in the well-documented growth arrest but also loss of culturability and overgrowth of the productive population using plasmid-deficient bacteria. The highest yields of soluble CheY∷GFP as judged from the fluorescence levels were achieved using very low concentrations of IPTG, which avoid growth arrest and loss of culturability postinduction. Optimal product yields were obtained with 8 μM IPTG, a concentration so low that insufficient T7 RNA polymerase accumulated to be detectable by Western blot analysis. The improved protocol was shown to be suitable for process scale-up and intensification. It is also applicable to the accumulation of an untagged heterologous protein, cytochrome c(2) from Neisseria gonorrhoeae, which requires both secretion and extensive post-translational modification.

© 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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