title: Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli.
abstract: We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach ("InFFact") made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli). In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment ("InFFact-GFP"), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting. In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner. In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS and Universités d'Aix-Marseille I and II, Case 932 Campus de Luminy, 163 Avenue de Luminy 13288 Marseille Cedex 09, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Coutard
authors.forename: Mathieu
authors.initials: M
authors.lastname: Gagnaire
authors.forename: Aude-Agnès
authors.initials: AA
authors.lastname: Guilhon
authors.forename: Marine
authors.initials: M
authors.lastname: Berro
authors.forename: Stéphane
authors.initials: S
authors.lastname: Canaan
authors.forename: Christophe
authors.initials: C
authors.lastname: Bignon
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins