Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening
文献类型：期刊论文
| 作者 | Cheng, DH; Shen, Q; Qian, J; Qian, Z; Ye, QZ |
| 刊名 | ACTA PHARMACOLOGICA SINICA
 |
| 出版日期 | 2002-02 |
| 卷号 | 23期号:2页码:143-151 |
| 关键词 | matrix metalloproteinases human macrophage elastase gene expression high-throughput screening |
| ISSN号 | 1671-4083 |
| 文献子类 | Article |
| 英文摘要 | AIM: To obtain a catalytically active human macrophage elastase catalytic domain (hMECD) and to establish an efficient high-throughput method for screening macrophage elastase inhibitors. METHODS: Catalytic domain of human macrophage elastase was expressed in E coli and characterized to establish a high-throughput screening assay using a colorimetric method. A set of 8560 pure compounds and mixtures were screened. RESULTS: We have constructed an efficient E coli system for this human protein expression, and them recombinant hMECD protein was purified to homogeneity using anion-exchange chromatography after in vitro refolding from inclusion bodies. The yield of active hMECD protein was 23 mg from one liter of E coli culture after purification. Calcium and zinc ions were required both in refolding and enzymatic activity, but high concentration of zinc inhibited the refolding and activity. The hMECD cleaved several synthetic substrates including a chromogenic thiopeptolide and fluorogenic peptides, with optimal activity around pH 8.0. Screening of 8560 compounds and mixtures led to identify 27 pure compounds and 14 natural products with inhibitory activity higher than 80 % at 20 mg/L. CONCLUSION - An efficient expression and purification method for hMECD protein has been established,, and the assay is effective, reliable, and fast in identifying the recombinant protein inhibitors. |
| WOS关键词 | MATRIX METALLOPROTEINASES ; ESCHERICHIA-COLI ; STROMELYSIN ; ACTIVATION ; MECHANISMS ; PROTEINS ; TUMOR |
| WOS研究方向 | Chemistry ; Pharmacology & Pharmacy |
| 语种 | 英语 |
| WOS记录号 | WOS:000173710300009 |
| 出版者 | ACTA PHARMACOLOGICA SINICA |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/274389]  |
| 专题 | 国家新药筛选中心 |
| 通讯作者 | Ye, QZ |
| 作者单位 | Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Mat Med, Natl Ctr Drug Screening, Shanghai 201203, Peoples R China |
| 推荐引用方式 GB/T 7714 | Cheng, DH,Shen, Q,Qian, J,et al. Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening[J]. ACTA PHARMACOLOGICA SINICA,2002,23(2):143-151. |
| APA | Cheng, DH,Shen, Q,Qian, J,Qian, Z,&Ye, QZ.(2002).Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening.ACTA PHARMACOLOGICA SINICA,23(2),143-151. |
| MLA | Cheng, DH,et al."Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening".ACTA PHARMACOLOGICA SINICA 23.2(2002):143-151. |
入库方式： OAI收割
来源：上海药物研究所
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