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

Abstract

Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C in ) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C out ) to C in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescencedetection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C out topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells

“…5b). However, we are not sure whether this stabilizing effect is caused by the directed interaction between lipid and the transporter or via changes in micelle structure [21]. Indeed, this was surprising since the stability of membrane proteins in native membranes is expected to be higher than in a detergent solubilized form.…”

“…Detergent solubilized membrane protein-GFP fusion is run on SDS-PAGE and intensity of GFP signal is flowed by exciting at 488 nm and emission at 535 nm [21]. We selected 20 different detergents from all three classes i.e.…”

Green Fluorescent Protein as a protein localization and topological reporter in mycobacteria

“…The pJB vector system consists of two vectors, pJB(-) and JB(+), engineered from the mycobacterial expression plasmid, pMV261 [10], and the E. coli expression plasmids, pWARF(+) or pWARF(-) [11]. This system allows for the mycobacterial expression under control of the p hsp60 promoter of proteins C-terminally-fused to the GFP and to a hexahistidine tag that may subsequently be easily purified from a Mycobacterium host.…”

“…pJB(+) has the HRV 3C protease cleavage site, followed by the transmembrane segment of glycophorin A, GFP and a C-terminal His 8 tag [Fig. 1A] [11]. Gene fusions in pJB(-) and JB(+) may be generated from the same PCR fragment.…”

“…Gene fusions in pJB(-) and JB(+) may be generated from the same PCR fragment. Based on earlier E. coli studies [11], it was expected that the addition of a single transmembrane domain from glycophorin A between the C-terminal fusion point of the protein of interest and the GFP in pJB(+) would allow membrane proteins with extracellular C-terminal fusions in mycobacteria to be converted to proteins with intracellular C-terminal fusions [Fig. 1B].…”