Open AccessExploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high‐quality recombinant proteins
Author(s) -
Sevastsyanovich Yanina,
Alfasi Sara,
Overton Tim,
Hall Richard,
Jones Jo,
Hewitt Christopher,
Cole Jeff
Publication year - 2009
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2009.01738.x
Subject(s) - green fluorescent protein , t7 rna polymerase , fusion protein , escherichia coli , lac operon , biology , recombinant dna , inclusion bodies , population , biochemistry , chemistry , microbiology and biotechnology , gene , demography , bacteriophage , sociology
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.