High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

Abstract

The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A 2 a receptor fused to a green fluorescent protein (A 2 aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A 2 aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A 2 aR and A 2 aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A 2 aR-GFP-His 10 . One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A 2 aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A 2 a-His 10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A 2 aR yet achieved from any heterologous expression system.

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

“…As evidence of the essential functions of these proteins, more than half of all drug targets have been shown to be transmembrane proteins, and the analysis of the interactions of transmembrane proteins and their ligands is one of the most promising avenues for the discovery of new drug candidates. As a means of producing sufficient amounts of transmembrane proteins for binding analyses, heterologous protein expression systems have been developed using Escherichia coli (10), yeast (16), insect, and mammalian (4) cells as hosts. Transmembrane proteins generally are expressed at low levels and are extremely hydrophobic, rendering the analysis of interactions with ligands very difficult.…”

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…J. 2012, 7, 620-634 www.biotechnology-journal.com GFP) followed by a tag optimal for purification (e.g., GFP-His 6 [160] or GFP-His 10 [161]). Tandem affinity purification is an approach where the protein of interest is fused in-frame to two affinity tags, generally separated by a protease cleavage site.…”

Progress toward heterologous expression of active G‐protein‐coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

“…Previously, we have shown that hA 2 aR is folded in an active, ligand-binding conformation in whole-cell assays, in membrane preparations, and once purified from the cells. 14 present in yeast cells, we would have expected to detect a minimum of 10% of the active complex (B max ) values measured from mammalian controls. Using this as the limit of detection suggests that active A 1 R levels were less than 5 pmol/mg (based on NECA), and for A 3 R, less than 0.2 pmol/mg (IB-MECA).…”

“…14,15,18 Each GPCR was subcloned directly into the pITy-MC-His 10 plasmid 14 and the pITy-MCeGFP-His 10 plasmid. Both vectors contain a multiple cloning (MC) site consisting of EagI, AatII, and SacII immediately following the Gal1-10 promotor and synthetic pre-pro N-terminal leader sequence.…”

“…Yeast have been successfully used for the heterologous expression of membrane proteins, specifically GPCRs. 12,13 Recent expression of the human adenosine A 2 a receptor (hA 2 aR) in S. cerevisiae has yielded active protein at greater than 10 mg/L of culture, which has facilitated its purification 14 and biophysical characterization (O'Malley, Naranjo, Lazarova, and Robinson, manuscript in preparation). However, under identical culture and expression conditions, the human neurokinin NK 1 (hNK 1 ) receptor fails to traffic to the plasma membrane in this system and does not display ligand-binding activity.…”