Recombinant G Protein-Coupled Receptor Expression in Saccharomyces cerevisiae for Protein Characterization

Blocker, Kory M.; Britton, Zachary T.; Naranjo, Andrea; McNeely, Patrick M.; Young, Carissa L.; Robinson, Anne S.

doi:10.1016/bs.mie.2014.12.025

Cited by 7 publications

(11 citation statements)

References 65 publications

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“…The full-length, wild type human A 2A receptor (A 2A R) was expressed in yeast BJ5464 cells, and membrane preparations were carried out as described in [23] and the Materials and Methods section. Samples of A 2A R were extracted and purified from cellular membranes using different detergent and lipid mixtures: DDM, CHAPS, CHS, and DHPC.…”

Section: Resultsmentioning

confidence: 99%

“…In order to improve protein purity and reproducibility between purifications, a previous purification protocol [11, 22] was adapted to employ membrane preparations instead of crude cell lysis [23]. Briefly, cell pellets were collected via centrifugation 24 hours post-induction.…”

Section: Methodsmentioning

confidence: 99%

“…For details regarding the purification protocol refer to [23]. For these studies, additional detergent and lipid mixtures were used as follows.…”

Section: Methodsmentioning

confidence: 99%

“…5.6 µM CPM was added to the protein sample while A 2A R was bound to Ni-NTA Superflow resin prior to elution (CPM was allowed to react with accessible free thiols, protected from light, on an end-over-end mixer at 4 °C overnight). The following day the protein was eluted from the Ni-NTA Superflow resin with 500 mM imidazole as described in [23]. …”

Section: Methodsmentioning

confidence: 99%

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Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

Naranjo

McNeely

Katsaras³

et al. 2016

Protein Expression and Purification

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The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoylsn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. The studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…For details regarding the purification protocol refer to [23]. For these studies, additional detergent and lipid mixtures were used as follows.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

Naranjo

McNeely

Katsaras³

et al. 2016

Protein Expression and Purification

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“…The purified proteins were desalted using extensive dialysis. The size and purity of each desalted protein were determined using 15% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 55 .…”

Section: Methodsmentioning

confidence: 99%

Citronellal perception and transmission by Anopheles gambiae s.s. (Diptera: Culicidae) females

Yang

et al. 2020

Sci Rep

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Anopheles gambiae s.s. is a key vector of Plasmodium parasites. Repellents, which may be a promising alternative to pesticides used to control malaria mosquitoes. Although citronellal is a known mosquito repellent, its repellency characteristics are largely unknown. Determining the specific odorant-binding proteins (OBPs) and odorant receptors (ORs) that detect and transfer the citronellal molecule in A. gambiae s.s. will help to define the mode of action of this compound. In this research, we assessed the repellent activity of citronellal in A. gambiae s.s. using a Y-tube olfactory meter, screened candidate citronellal-binding OBPs and ORs using reverse molecular docking, clarified the binding properties of predicted proteins for citronellal using fluorescence competition binding assay. Results showed that citronellal had a dosage effect on repelling A. gambiae s.s.. The 50% repellent rate was determined to be 4.02 nmol. Results of simulated molecular docking showed that the only proteins that bound tightly with citronellal were AgamOBP4 and AgamORC7. Fluorescence competitive binding assays confirmed the simulations. This research determined that citronellal was captured by AgamOBP4 and transmitted to AgamORC7 in A. gambiae s.s.. Our study will be beneficial in the further understanding the repellent mechanism of citronellal against A. gambiae s.s..

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Using Fluorescence Anisotropy for Ligand Binding Kinetics of Membrane Proteins

Swonger

Robinson

2018

CP Protein Science

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Determining ligand binding kinetics provides an indirect route to probe the functional capabilities of the binding pocket of a membrane protein receptor. Presented in this unit are four ligand-binding protocols that provide data useful for characterizing membrane proteins, including equilibrium binding, thermostability, competitive ligand binding, and kinetic ligand binding. These techniques use fluorescence anisotropy, which is safer, less costly, and simpler to execute than radioactive ligand binding. Each protocol may be used on its own or in combination with others to quantify a number of ligand binding constants. © 2018 by John Wiley & Sons, Inc.

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Recombinant G Protein-Coupled Receptor Expression in Saccharomyces cerevisiae for Protein Characterization

Cited by 7 publications

References 65 publications

Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

Citronellal perception and transmission by Anopheles gambiae s.s. (Diptera: Culicidae) females

Using Fluorescence Anisotropy for Ligand Binding Kinetics of Membrane Proteins

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