High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1

Ren, Hui; Yu, Daoyong; Ge, Baosheng; Cook, Brian L.; Xu, Zhinan; Zhang, Shuguang

doi:10.1371/journal.pone.0004509

Cited by 75 publications

(78 citation statements)

References 76 publications

(113 reference statements)

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“…33 Recently, it has been shown that FC-13 to FC-16 detergents could efficiently solubilize IBs formed by the human tachykinin G-protein coupled receptor NK1, expressed in E. coli 34 as well as several 7-transmembrane G-protein coupled chemokine receptors (also expressed in E. coli). 35 Thus, we propose that FC-12 (or longer chain FCs) and the protocol reported here could also be suitable for bacterial expression and purification of N-terminal cytosolic tails of all other a-subunit isoforms of V-ATPase.…”

Section: Discussionmentioning

confidence: 90%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

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V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N ) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N 1-402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINEV R 12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N 1-402 in NDSB-256 was successfully used in pull-down and BIAcore TM protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N 1-402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called ''cytosolic'' tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N 1-402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

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Section: Discussionmentioning

confidence: 90%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

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“…CD was used to verify that the cell-free produced receptors were properly folded, and that the peptide surfactants did not significantly affect the receptors' structures. In these experiments, the GPCRs were purified with fos-choline-14 (FC-14) because the peptide CD signal interferes with and overwhelms the receptor signals, and also because FC-14 has successfully been used with diverse GPCRs (21)(22)(23)(24). Fig.…”

Section: Resultsmentioning

confidence: 99%

Peptide surfactants for cell-free production of functional G protein-coupled receptors

Corin

Philipp

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Two major bottlenecks in elucidating the structure and function of membrane proteins are the difficulty of producing large quantities of functional receptors, and stabilizing them for a sufficient period of time. Selecting the right surfactant is thus crucial. Here we report using peptide surfactants in commercial Escherichia coli cell-free systems to rapidly produce milligram quantities of soluble G protein-coupled receptors (GPCRs). These include the human formyl peptide receptor, human trace amine-associated receptor, and two olfactory receptors. The GPCRs expressed in the presence of the peptide surfactants were soluble and had α-helical secondary structures, suggesting that they were properly folded. Microscale thermophoresis measurements showed that one olfactory receptor expressed using peptide surfactants bound its known ligand heptanal (molecular weight 114.18). These short and simple peptide surfactants may be able to facilitate the rapid production of GPCRs, or even other membrane proteins, for structure and function studies.in vitro translation | label-free M embrane proteins play vital roles in all living systems. Approximately 30% of genes in almost all sequenced genomes code for membrane proteins (1-3). However, our detailed understanding of membrane protein structure and function lags far behind that of soluble proteins. Indeed, as of April 2011, there are over 72,000 structures in the Protein Data Bank. Of these, only 280 are unique membrane proteins, and only six are unique G protein-coupled receptors (GPCRs). This surprising disparity is due to bottlenecks at nearly every stage of experimentation, from large-scale membrane protein production to X-ray crystal diffraction.Recent advances have overcome several bottlenecks in studying membrane proteins. Commercial development of nanoliter drop-setting robots and a wide variety of kits have made crystal screening less laborious. The development of worldwide accessible synchrotron facilities, and microfocus beamlines capable of collecting data from crystals <10-60 μm, have overcome the bottlenecks in data collection. Likewise, rapid advancements in computing power and an increase in open access software development have made the determination of structures a much less daunting task. However, inexpensive large-scale production of soluble and nonaggregated membrane proteins still remains a formidable challenge. Likewise, systematic surfactant screens still remain one of the most time-consuming and expensive experimental tasks. To overcome these bottlenecks, the discovery or invention of simple and broadly useful surfactants is crucial.Several cell-based membrane protein production systems have been developed, but they are costly and require months to generate sufficient quantities of protein. Commercial cell-free systems are alternative methods of producing membrane proteins. However, to produce nonaggregated membrane proteins, optimal selection of surfactants is critical: The newly produced membrane proteins must not only fold correctly, they must al...

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“…The rho1D4 elution peptide Ac-TETSQVAPA-CONH 2 was synthesized by CBC Scientific. Rho1D4-Sepharose immunoaffinity purification has been described (5,7,25,26).…”

Section: Methodsmentioning

confidence: 99%

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

Cook

Steuerwald

Kaiser

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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131

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Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be Ϸ50% ␣-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices. BiacoreA100 ͉ detergent screen ͉ fos-choline 14 ͉ G protein-coupled receptor purification ͉ membrane protein A nimal noses have evolved the ability to rapidly detect a seemingly infinite array of odors at minute concentrations. The basis of this sensitivity are the olfactory (smell) receptors, a large class of G protein-coupled receptors (GPCRs) that function together combinatorially to allow discrimination between a wide range of volatile and soluble molecules (1, 2). As GPCRs, all olfactory receptors (ORs) are integral membrane proteins with 7 predicted transmembrane domains. To date, crystal structures exist for only 5 GPCR proteins (3). Despite the fact that ORs represent the largest class of known membrane proteins, no detailed structure exists for any OR, because the major obstacle to structural and functional studies on membrane proteins is the notorious difficulty involved in expressing and purifying the large quantities of receptor protein sample required for techniques such as X-ray crystallography. The first crucial step to enable such pivotal biochemical and structural analyses is to engineer systems with the capacity to produce and purify milligram quantities of an OR. hOR17-4 (alternately known as OR1D2) is of particular interest because, in addition to olfactory neurons, it is exp...

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High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1

Cited by 75 publications

References 76 publications

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

Peptide surfactants for cell-free production of functional G protein-coupled receptors

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

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