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Title: Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high quality recombinant proteins
Authors: Sevastsyanovich, Yanina
Alfasi, Sara
Overton, Tim
Hall, Richard
Jones, Jo
Hewitt, Christopher J.
Cole, Jeff
Keywords: Recombinant protein production
Inclusion bodies
Gonococcal 1 cytochrome c2
Heat shock
General stress response
Green fluorescent protein
Flow cytometry
Issue Date: 2009
Publisher: © Federation of European Microbiological Societies / Blackwell Publishing Ltd
Citation: SEVASTSYANOVICH, Y. ... et al, 2009. Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high quality recombinant proteins. FEMS Microbiology Letters, 299 (1), pp. 86-94.
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.
Description: This research letter was submitted for publication in the journal, FEMS Microbiology Letters and the definitive version is available from: http://www3.interscience.wiley.com/journal/118512081/home
Version: Submitted for publication
DOI: 10.1111/j.1574-6968.2009.01738.x
URI: https://dspace.lboro.ac.uk/2134/5759
Publisher Link: http://www3.interscience.wiley.com/journal/118512081/home
ISSN: 0378-1097
Appears in Collections:Published Articles (Chemical Engineering)

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