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. 2007 Aug;159(2):166-78.
doi: 10.1016/j.jsb.2007.05.001. Epub 2007 May 16.

High-level Expression in Saccharomyces Cerevisiae Enables Isolation and Spectroscopic Characterization of Functional Human Adenosine A2a Receptor

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High-level Expression in Saccharomyces Cerevisiae Enables Isolation and Spectroscopic Characterization of Functional Human Adenosine A2a Receptor

Michelle A O'Malley et al. J Struct Biol. .
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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 (approximately 4mg/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-beta-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+/-2mg/L of culture, with ligand-binding yields of 1mg/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.


Figure 1
Figure 1. Western blot screening reveals the relative total expression levels of A2aR-His10 in transformed BJ5464 cells
Lanes labeled 1–11 represent different isolated clones that have been transformed with pITy-A2aR-His10 DNA and induced for 25 hours of expression. BJAG represents BJ A2aRGFP high-expressing cells (Niebauer and Robinson, 2006) also induced for 25 hours of expression. BJ represents the parental yeast strain, BJ5464 cells which underwent electroporation in the absence of pITy-A2aR-His10 DNA, treated under the same expression conditions. Rabbit anti-A2aR primary antibody was used to detect A2aR for all samples. Arrows indicate the migrations of A2aR-His10 monomer and dimer, as well as A2aR-GFP monomer and dimer bands.
Figure 2
Figure 2. Immobilized metal affinity chromatography enables purification of A2aR from batch culture using two lysis methods
Coomassie-stained 12% SDS-PAGE gels illustrate the purification process. (A) A2aR-His10 was eluted using 500 mM imidazole as recovered using the bead-vortexing lysis method. (B) A2aR-His10 was also purified using the membrane solubilization method. Similar results were obtained for the purification using both methods. All samples were solubilized in purification buffer containing 2% DDM, 1% CHAPS, and 0.2% CHS and supplemented with protease inhibitors and 1 mM PMSF. Wash steps were carried out in purification buffer containing 0.1% DDM, 0.1% CHAPS, and 0.02% CHS supplemented with protease inhibitors and 1 mM PMSF. This cell/surfactant flow-through (FT) denotes proteins that were present in the crude lysate, and did not bind to the resin. 20 mM–50 mM lanes show proteins which were removed from the resin during washes with low-concentration imidazole. The ladder is See-BluePlus2 protein molecular weight standard, with molecular weights indicated. A2aR-His10 monomer is denoted with the arrows.
Figure 3
Figure 3. DDM/CHAPS/CHS solubilized A2aR constitutes active, functional protein whereas DDM solubilized A2aR is inactive
Saturation ligand binding on purified A2aR solubilized in DDM/CHAPS/CHS (circles, solid) and DDM (diamonds, dashed) using 3H-CGS-21,680 ligand. Points indicate experimentally determined data, while lines indicate the best fit to a single-site model, where Kd for the active receptor was determined to be 66 +/− 4 nM. Bmax was 92 +/− 21 pmol/mg from the best fit of this data to a single-site binding model.
Figure 4
Figure 4. Xanthine affinity chromatography shows full-length A2aR is active in ligand-binding
Silver-stained 12% SDS-PAGE gel tracks purification of an IMAC purified population of A2aGFPHis10 through xanthine ligand affinity chromatography. The IMAC elute lane denotes protein loaded onto the XAC column which has been desalted and diluted in purification buffer containing 0.1% DDM/0.1% CHAPS/0.02% CHS as described in Materials and Methods. XAC wash lanes represent undiluted samples from 4 mL fractions collected as unbound material from the column. XAC elute lanes represent undiluted samples collected from 4 mL fractions and show active A2aGFPHis10 that was eluted from the column with 20mM theophylline.
Figure 5
Figure 5. Purified A2aR exhibits alpha-helical structure in the presence of CHS/CHAPS
CD spectrum of purified A2aR-His10 in DDM/CHAPS/CHS micelles (solid) and purified A2aR-His10 in DDM micelles without CHAPS/CHS (dotted). Both spectra are representative of three separate samples.
Figure 6
Figure 6. Fluorescence spectroscopy verifies that solubilized A2aR-His 10 in DDM both with CHS/CHAPS and without is properly incorporated into micelles
Intrinsic fluorescence spectra for A2aR-His10 in DDM micelles without CHAPS/CHS (dashed) and with CHAPS/CHS (solid). Fluorescence maxima are listed on the spectra.

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