A scalable, GFP‐based pipeline for membrane protein overexpression screening and purification

Abstract

We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.Keywords: Escherichia coli; membrane protein overexpression; membrane protein isolation; membrane protein characterization; GFP Membrane proteins (MPs) account for 20%-25% of all open reading frames in sequenced genomes, and fulfill a wide range of central functions in the cell (Wallin and von Heijne 1998). However, our knowledge of this important class of proteins is still poor, mainly because of a lack of generally applicable approaches to the overexpression and purification steps that precede functional and structural analysis. Novel approaches in these areas are required to facilitate and speed up MP research.The bacterium Escherichia coli is still the most widely used vehicle for MP overexpression. Overexpression in the cytoplasmic membrane is preferred to overexpression in inclusion bodies, since the isolation of functional MPs from the membrane is usually more successful than refolding from inclusion bodies (Drew et al. 2003). Green fluorescent protein (GFP) fusions can be used to facilitate the monitoring of MP overexpression in the cytoplasmic membrane (Drew et al. 2001). If the fusion protein ends up in inclusion bodies, GFP does not fold and is therefore not fluorescent; in contrast, if the fusion is expressed in the cytoplasmic membrane, GFP folds properly and is fluorescent. GFP is only fluorescent in the cytoplasm of Escherichia coli (Drew et al. 2002), which means that GFP-based screens work only for MPs that have their C terminus located in the cytoplasm. Recently, nearly all E. coli cytoplasmic MPs were fused to GFP for a membrane proteome topology screen (Daley et al. 2005). Approximately 80% of all E. coli cytoplasmic MPs have a cytoplasmic C terminus, and thus GFP can be used to monitor the overexpression levels of the majority of E. coli MPs (Daley et al. 2005).Here, we present a generic pipeline for rapid overexpression screening, detergent extraction, and purification of MPs based on a simple MP-GFP fusion approach. We show that milligram amounts of pure functional MP can Reprint requests to: Jan-Willem de Gier, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; e-mail: degier@dbb.su.se; fax: +46-8-153679.Article published online ahead of print. Article and publication date are at

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

“…As the C-terminal GFP folds and becomes fluorescent only if the upstream membrane protein integrates into the membrane, the resultant fluorescence is a fast and accurate measure of membrane-integrated expression 1 . Fluorescence is easy to measure directly in liquid culture, standard SDS-gels and detergentsolubilized membranes 2,3 . Detergent-solubilized membranes can also be subjected to fluorescence size-exclusion chromatography (FSEC) to measure the 'monodispersity' of the sample 4 .…”

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

“…Building upon previously described reports using GFP as a reporter for monitoring expression and purification, [3][4][5][6][7][8][9][10][14][15][16] we established a comprehensive, streamlined approach for confirming topology, optimizing expression, and screening stable protein-detergent complexes for both C in and C out membrane proteins. The pWarf vector system consists of two vectors, pWarf(À) and pWarf(þ), that were constructed by modifying the pET28 vector described in Drew et al 9 (Supporting Information Fig.…”

“…The pWarf vector system consists of two vectors, pWarf(À) and pWarf(þ), that were constructed by modifying the pET28 vector described in Drew et al 9 (Supporting Information Fig. 1).…”

“…18 All genes were successfully cloned into both vectors and subjected to expression screening and optimization. Whole-cell GFP fluorescence was monitored to quantify expression levels based on a method previously reported, 9 where the whole-cell GFP fluorescence was directly proportional to membrane protein yield.…”

“…Several studies have employed GFP as a reporter for membrane protein expression and detergent selection, [3][4][5][6]9,10,14,15 but the technology has largely been restricted to proteins with intracellular C-termini (C in ) due to the GFP's inability to mature when localized to the E. coli periplasm. 11 We have demonstrated that Glycophorin A (GpA)-a single spanning transmembrane helix-effectively converts C out membrane proteins to become C in proteins where the GFP can properly fold and fluoresce.…”