Evidence map›Paper›PMID 26073323›Full record

ArticleActa pharmacologica Sinica2015

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

Hao Cheng, Chen Fan, Si-wei Zhang, Zhong-shan Wu, Zhi-cheng Cui, Karsten Melcher, Cheng-hai Zhang, Yi Jiang, Yao Cong, H Eric Xu

Open access · bronzeAbstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Acta pharmacologica Sinica, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.

0numbers the graph read from it
0cells of the map it votes in
7citing papers in PubMed
0.6field-weighted citation impact, top 32% of its field
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

7 citing papers in PubMed, 8 citations in OpenAlex.

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  3. ConoMode, a database for conopeptide binding modes.Database : the journal of biological databases and curation · 2020
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  4. Article
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  7. Review
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

10 authors at 3 institutions in 2 countries.

Hao Cheng1] VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China [2] National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China.
Chen FanNational Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China.
Si-wei ZhangVARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Zhong-shan Wu1] VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China [2] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.
Zhi-cheng CuiNational Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China.
Karsten MelcherLaboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
Cheng-hai ZhangVARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Yi JiangVARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Yao CongNational Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China.
H Eric Xu1] VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China [2] Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
Chinese Academy of Sciences · CNShanghai Institute of Materia Medica · CNVan Andel Institute · US

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

aimTo report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target.

methodsα7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis.

resultsExpression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel.

conclusionWe have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.

Indexed as

alpha7 Nicotinic Acetylcholine ReceptorAmino Acid SequenceAnimalsChromatography, AffinityCloning, MolecularCrystallizationCrystallography, X-RayHEK293 CellsHumansModels, MolecularMolecular Sequence DataProtein StabilitySequence AlignmentTemperaturealpha7 Nicotinic Acetylcholine Receptor

Identifiers

PMID26073323
PMCPMC4564878
OpenAlexW1808858027

What OpenQuestion holds

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Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.