High yield and efficient expression and purification of the human 5-HT3A receptor
文献类型：期刊论文
| 作者 | Wu, Zhong-shan2,3; Cui, Zhi-cheng4; Cheng, Hao3; Fan, Chen4; Melcher, Karsten1; Jiang, Yi3 ; Zhang, Cheng-hai3; Jiang, Hua-liang5; Cong, Yao3; Liu, Qian2
|
| 刊名 | ACTA PHARMACOLOGICA SINICA
 |
| 出版日期 | 2015-08 |
| 卷号 | 36期号:8页码:1024-1032 |
| ISSN号 | 1671-4083 |
| 关键词 | human 5-HT3A receptor BacMam expression and purification negative stain electron microscopy receptor structure |
| DOI | 10.1038/aps.2015.35 |
| 文献子类 | Article |
| 英文摘要 | Aim: To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies. Methods: Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8xHis tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM). Results: The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure. Conclusion: With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor. |
| WOS关键词 | GATED ION-CHANNEL ; CYS-LOOP RECEPTOR ; X-RAY-STRUCTURE ; STRUCTURAL BASIS ; SYSTEM ; PHARMACOLOGY ; RECOGNITION ; CONDUCTANCE ; ANTAGONISTS ; PREVENTION |
| 资助项目 | Jay and Betty Van Andel Foundation[00000000] ; Ministry of Science and Technology (China)[2012ZX09301001] ; Ministry of Science and Technology (China)[2012CB910403] ; Ministry of Science and Technology (China)[2013CB910600] ; Ministry of Science and Technology (China)[XDB08020303] ; Ministry of Science and Technology (China)[2013ZX09507001] ; Ministry of Science and Technology (China)[XDB08030201] ; Ministry of Science and Technology (China)[13JC1406300] ; Ministry of Science and Technology (China)[31222016] ; Ministry of Science and Technology (China)[31270771] ; Shanghai Science and Technology Committee[13JC1406300] ; Shanghai Science and Technology Committee[13ZR1447600] |
| WOS研究方向 | Chemistry ; Pharmacology & Pharmacy |
| 语种 | 英语 |
| CSCD记录号 | CSCD:5487151 |
| 出版者 | ACTA PHARMACOLOGICA SINICA |
| WOS记录号 | WOS:000358934000013 |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/276454]  |
| 专题 | 药物靶标结构与功能中心 中科院受体结构与功能重点实验室 新药研究国家重点实验室 药物发现与设计中心 |
| 通讯作者 | Liu, Qian |
| 作者单位 | 1.Van Andel Res Inst, Lab Struct Sci, Grand Rapids, MI 49503 USA; 2.Huazhong Univ Sci & Technol, Britton Chance Ctr Biomed Photon, Wuhan Natl Lab Optoelect, Wuhan 430074, Peoples R China; 3.Chinese Acad Sci, VARI SIMM Ctr, Ctr Struct & Funct Drug Targets, CAS Key Lab Receptor Res,Shanghai Inst Mat Med, Shanghai 201203, Peoples R China; 4.Chinese Acad Sci, Nat Ctr Prot Sci Shanghai, Inst Biochem & Cell Biol, Shanghai Inst Biol Sci,State Key Lab Mol Biol, Shanghai 201210, Peoples R China; 5.Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Shanghai 201203, Peoples R China |
| 推荐引用方式 GB/T 7714 | Wu, Zhong-shan,Cui, Zhi-cheng,Cheng, Hao,et al. High yield and efficient expression and purification of the human 5-HT3A receptor[J]. ACTA PHARMACOLOGICA SINICA,2015,36(8):1024-1032. |
| APA | Wu, Zhong-shan.,Cui, Zhi-cheng.,Cheng, Hao.,Fan, Chen.,Melcher, Karsten.,...&Xu, H. Eric.(2015).High yield and efficient expression and purification of the human 5-HT3A receptor.ACTA PHARMACOLOGICA SINICA,36(8),1024-1032. |
| MLA | Wu, Zhong-shan,et al."High yield and efficient expression and purification of the human 5-HT3A receptor".ACTA PHARMACOLOGICA SINICA 36.8(2015):1024-1032. |
入库方式： OAI收割
来源：上海药物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。