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

Authors


  • Editor: Diethard Mattanovich

Correspondence: Jeff Cole, School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK. Tel.: +44 121 414 5440; fax: +44 121 414 5495; e-mail: j.a.cole@bham.ac.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 c2 from Neisseria gonorrhoeae, which requires both secretion and extensive post-translational modification.

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