title: Assessment of membrane protein expression and stability using a split green fluorescent protein reporter.
abstract: Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP(11)) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP(1-10)). The signal obtained after sequential expression of SteT (l-serine/l-threonine exchanger of Bacillus subtilis) fused to GFP(11) followed by GFP(1-10) specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT-GFP(11) with purified GFP(1-10) was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT-GFP(11) following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.
authors.affiliation: Institute for Research in Biomedicine, 08028 Barcelona, Spain.
authors.forename: Arturo
authors.initials: A
authors.lastname: Rodríguez-Banqueri
authors.forename: Lukasz
authors.initials: L
authors.lastname: Kowalczyk
authors.forename: Manuel
authors.initials: M
authors.lastname: Palacín
authors.forename: José Luis
authors.initials: JL
authors.lastname: Vázquez-Ibar
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018360:Crystallography, X-Ray; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins