Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study
文献类型：期刊论文
| 作者 | Liu, Jiafu1; Lin, Kejiang1; Guo, Chenyun2; Gao, Hongchang2; Yao, Yihe2; Lin, Donghai1,2 |
| 刊名 | ACTA BIOCHIMICA ET BIOPHYSICA SINICA
 |
| 出版日期 | 2008-06 |
| 卷号 | 40期号:6页码:489-496 |
| 关键词 | lipocalin-type prostaglandin D synthase mutant HSQC heteronuclear NMR |
| ISSN号 | 1672-9145 |
| DOI | 10.1111/j.1745-7270.2008.00426.x |
| 文献子类 | Article |
| 英文摘要 | Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH(2) to produce PGD(2). Three cysteine residues, Cys(65), Cys(89), and Cys(186) in L-PGDS, are conserved among all species, of which Cys(89) and Cys(186) residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled rat L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure. |
| WOS关键词 | CIRCULAR-DICHROISM ; PROTEINS ; NMR ; BINDING ; INTERMEDIATE ; SYSTEM ; ENZYME ; CAVITY |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biophysics |
| 语种 | 英语 |
| CSCD记录号 | CSCD:3301262 |
| WOS记录号 | WOS:000256851700004 |
| 出版者 | OXFORD UNIV PRESS |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/272897]  |
| 专题 | 分析化学研究室 |
| 通讯作者 | Lin, Donghai |
| 作者单位 | 1.China Pharmaceut Univ, Sch Life Sci & Technol, Nanjing 210009, Peoples R China; 2.Chinese Acad Sci, Shanghai Inst Biol Sci, Shanghai Inst Mat Med, Shanghai 201203, Peoples R China |
| 推荐引用方式 GB/T 7714 | Liu, Jiafu,Lin, Kejiang,Guo, Chenyun,et al. Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study[J]. ACTA BIOCHIMICA ET BIOPHYSICA SINICA,2008,40(6):489-496. |
| APA | Liu, Jiafu,Lin, Kejiang,Guo, Chenyun,Gao, Hongchang,Yao, Yihe,&Lin, Donghai.(2008).Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study.ACTA BIOCHIMICA ET BIOPHYSICA SINICA,40(6),489-496. |
| MLA | Liu, Jiafu,et al."Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study".ACTA BIOCHIMICA ET BIOPHYSICA SINICA 40.6(2008):489-496. |
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。