Purification and characterization of recombinant human endothelin receptor type A

Lee, Kwang-Kyu; Jung, Yuna; Lee, Jae Youl; Lee, Won-Kyu; Lim, Dongbin; Yu, Yeon Gyu

doi:10.1016/j.pep.2012.04.011

Cited by 19 publications

(25 citation statements)

References 27 publications

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“…The purified P9-5-HT 3A ran as a 56-kDa protein in SDS–PAGE, which was slightly lower than the calculated size (67 kDa). Membrane proteins such as GPCRs often migrate in SDS–PAGE faster than their actual size [17]. The identity of the purified protein as P9-5-HT 3A was further confirmed by western blotting using anti-His6 antibody (data not shown).…”

Section: Resultsmentioning

confidence: 92%

“…Previously, we have reported that human ET A could be highly expressed in the membrane fraction in E. coli when ET A was expressed as a P9 fusion protein [17]. To achieve this, the P9 protein was fused to the N-terminus of the wild-type or W178A mutant 5-HT 3A along with a His6 tag at the C-terminus, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The expression of membrane proteins in bacteria has been accomplished using specialized fusion partners such as Mistic [14], green fluorescent protein (GFP) [15], or maltose-binding protein and thioredoxin [16]. Recently, we had successively expressed human endothelin receptor type A (ET A ), a member of the G-protein coupled receptors, in Escherichia coli using the P9 protein of bacteriophage phi6 as a fusion partner of ET A [17]. Most of the expressed P9-ET A was located in the membrane fraction, and it could be purified to homogeneity with high yield, suggesting that the P9 protein could be used as a fusion partner to assist in high-level expressions of other membrane proteins such as 5-HT 3A in the membrane fraction of E. coli .…”

Section: Introductionmentioning

confidence: 99%

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Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes

Shin

Jung

et al. 2013

Protein Expression and Purification

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Human serotonin receptor 3A (5-HT3A) is a ligand-gated ion channel regulated by serotonin. A fusion protein (P9-5-HT3A) of 5-HT3A with the P9 protein, a major envelope protein of bacteriophage phi6, was highly expressed in the membrane fraction of Escherichia coli, and the expressed protein was purified to homogeneity using an affinity chromatography. P9-5-HT3A was observed as mixed oligomers in detergents. The purified P9-5-HT3A was efficiently reconstituted into proteoliposomes, and the serotonin-dependent ion-channel activity of P9-5-HT3A was observed by measuring the increased fluorescence of Fluo-3 attributed to the formation of a complex with the Ca2+ ions released from the proteoliposomes. Alanine substitution for Trp178 of 5-HT3A abolished the serotonin-dependent ion-channel activity, confirming the importance of Trp178 as a ligand-binding site. Furthermore, the ion-channel activity of the reconstituted P9-5-HT3A was effectively blocked by treatment with ondansetron, an antagonist of 5-HT3A. The bacterial expression system of human 5-HT3A and the proteoliposomes reconstituted with 5-HT3A would provide biophysical and structural analyses of 5-HT3A.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes

Shin

Jung

et al. 2013

Protein Expression and Purification

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“…As shown in Figure 4(C), specific binding of biotinylated ET-1 to the APG:P9-ET A was observed and had an apparent equilibrium dissociation constant (K D ) of 60 nM, which is comparable to the reported K D using a purified ET A . 20 These results indicated that APG stabilized BR and ET A in their native conformations.…”

Section: Apg Solubilizes Escherichia Coli Membrane Proteins and Stabimentioning

confidence: 84%

“…A fusion protein of ET A and P9, an envelope protein of Pseudomonas phage phi6, was expressed in E. coli and purified as previously described. 20 The fusion protein of P9 and ET A (P9-ET A ) in sarkosyl or n-dodecyl-b-D-maltoside (DDM) was found to be poly-dispersed in gel-filtration chromatography [ Fig. 4(A), green and blue lines, respectively].…”

Section: Apg Solubilizes Escherichia Coli Membrane Proteins and Stabimentioning

confidence: 99%

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

Han

Lee

et al. 2014

Protein Science

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Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-c-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ET A ) in their active conformations. Furthermore, ET A in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.

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Overexpression and Functional Stabilization of Recombinant Human Lysophosphatidic Acid Receptor 1 Using an Amphiphatic Polymer

Han

Baek

Lee

et al. 2017

Bulletin Korean Chem Soc

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Human lysophosphatidic acid receptor 1 (LPA1 ) is a G‐protein coupled receptor that mediates various biological functions such as proliferation, platelet aggregation, smooth muscle contraction, and tumor cell invasion. For dissection of the molecular function of LPA1 , a recombinant LPA1 was overexpressed in Escherichia coli membrane fractions and purified to homogeneity by single affinity chromatography. The purified LPA1 was stabilized with an amphiphilic polymer that was synthesized by the coupling of octylamine, glucosamine, and diethylaminoproylamine at the carboxylic groups of poly‐γ‐glutamic acid. The complex of purified LPA1 and amphiphilic polymer showed a monodisperse oligomer and specific binding to LPA with apparent Ki values of 30 μM. Compared with the Gs protein, it also showed selective binding to the alpha subunit of the Gi protein. These results indicate that recombinant LPA1 in an amphiphilic polymer complex has an active conformation for interaction with ligands and G‐proteins.

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Purification and characterization of recombinant human endothelin receptor type A

Cited by 19 publications

References 27 publications

Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes

Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

Overexpression and Functional Stabilization of Recombinant Human Lysophosphatidic Acid Receptor 1 Using an Amphiphatic Polymer

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