Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins |
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Authors: | Yanina Sevastsyanovich Sara Alfasi Tim Overton Richard Hall Jo Jones Christopher Hewitt & Jeff Cole |
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Institution: | School of Biosciences, University of Birmingham, Birmingham, UK;;School of Chemical Engineering, University of Birmingham, Birmingham, UK;;GlaxoSmithKline Research and Development, Herts, UK;and;Department of Chemical Engineering, University of Loughborough, Leicestershire, UK |
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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 c2 from Neisseria gonorrhoeae , which requires both secretion and extensive post-translational modification. |
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Keywords: | recombinant protein production inclusion bodies gonococcal cytochrome c2 general stress response green fluorescent protein flow cytometry |
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