Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins
2009
Sevastsyanovich, Yanina | Alfasi, Sara | Overton, Tim | Hall, Richard | Jones, Jo | Hewitt, Christopher | Cole, Jeff
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.
Show more [+] Less [-]AGROVOC Keywords
Bibliographic information
This bibliographic record has been provided by National Agricultural Library