Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

Abstract

We present an example of expression and purification of a biologically active G protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR α-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 μg of purified α-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified α-factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the α-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

Changes in Conformation at the Cytoplasmic Ends of the Fifth and Sixth Transmembrane Helices of a Yeast G Protein-Coupled Receptor in Response to Ligand Binding

“…Similar results are obtained with an antibody generated against the N-terminus of Ste2p. Furthermore, purified Ste2p confirmed by MALDI-TOF and nanospray MS exhibits a 100 kDa at the same position we have attributed to the Ste2p dimer (54). Most importantly, the major conclusion of this paper is that the results indicate that the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding.…”

Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2

“…Although the Rho-tag has been previously used for isolation of several other recombinant GPCR expressed in mammalian cells [6, 7, 24], yeast [25, 26] and in a cell-free systems [27], the usefulness of this tag for recombinant expression of membrane receptors in E. coli had not been reported yet. We observed that the C-terminal location of the Rho-tag in the fusion construct is obligatory to ensure its accessibility for interaction with specific 1D4 antibody while an appropriate spacer (4 amino acid residues or longer) introduced between the C-terminus of the receptor and the Rho- tag is necessary for high expression levels.…”

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

“…Recent advances in the expression and purification of membrane proteins have been described for various expression hosts, for example: Escherichia coli (Drew et al 2003(Drew et al , 2005Grisshammer et al 2005), yeast (Wedekind et al 2006;Lee et al 2007), insect cells (Massotte 2003), mammalian cells (Yin et al 2005;Werner et al 2008) and cell-free systems (Klammt et al 2007). However, from approximately 1000 known GPCRs, only five high-resolution 3-D structures of two distinct receptor types have been reported: bovine rhodopsin (Palczewski et al 2000) and opsin (Park et al 2008), squid rhodopsin (Murakami and Kouyama 2008), the human b2-adrenergic receptor Rosenbaum et al 2007) and the turkey b1-adrenergic receptor (Warne et al 2008).…”