Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening

doi:10.1016/S1046-5928(02)00530-2

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	Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening
	Cheng, DH; Shen, Q; Nan, FJ; Qian, Z; Ye, QZ
刊名	PROTEIN EXPRESSION AND PURIFICATION
	2003-01
卷号	27 期号:1 页码:63-74
关键词	matrix metalloproteinase human gelatinase A fibronectin catalytic domain gene expression high-throughput screening
ISSN号	1046-5928
DOI	10.1016/S1046-5928(02)00530-2
文献子类	Article
英文摘要	Gelatinase A represents an attractive therapeutic target for cancer invasion and metastasis. In order to screen for gelatinase A inhibitors, we have cloned, overexpressed in a bacterial system, and purified the catalytic domain of human gelatinase A with (GaCDfn) or without (GaCD) fibronectin-like insert. GaCDfn and GaCD were purified to homogeneity and refolded in vitro. GaCDfn was refolded to a stable and active form in the presence of calcium and zinc ions. GaCD was refolded through direct dialysis against Tris-HCl (pH 7.5) buffer without calcium and zinc ions. GaCD is unstable in the presence of calcium and zinc ions. The enzymatic activities of GaCDfn and GaCD require calcium and zinc ions, but high concentration of zinc and calcium ions inhibited the activities. The GaCDfn and GaCD cleaved several synthetic substrates including a chromogenic thiopeptolide (TPL) and fluorogenic peptides with optimal activity around pH 7.5. Moreover, GaCDfn and GaCD cleave gelatin and collagen VII and display similar cleavage patterns on the gel, but the digestion rate of these protein substrates by GaCD is apparently slower than GaCDfn. EDTA, 1,10-phenanthroline, and reference inhibitors potently blocked GaCDfn and GaCD enzymatic activities. A set of 3596 compounds from our center collection were screened by using GaCDfn and GaCD to cleave TPL. Further analysis by using MMP inhibitors indicated there is a correlation between IC50 values on GaCDfn and GaCD. A few compounds with selectivity toward gelatinase A catalytic domain were identified for structure modification. (C) 2002 Elsevier Science (USA). All rights reserved.
WOS关键词	MATRIX METALLOPROTEINASES ; TISSUE INHIBITOR ; HUMAN-SKIN ; FAMILY ; IDENTIFICATION ; SPECIFICITY ; ACTIVATION ; COLLAGEN ; PROTEIN ; BINDING
WOS研究方向	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
语种	英语
出版者	ACADEMIC PRESS INC ELSEVIER SCIENCE
WOS记录号	WOS:000180678700009
内容类型	期刊论文
源URL	[http://119.78.100.183/handle/2S10ELR8/274290]
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,Nan, FJ,et al. Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening[J]. PROTEIN EXPRESSION AND PURIFICATION,2003,27(1):63-74.
APA	Cheng, DH,Shen, Q,Nan, FJ,Qian, Z,&Ye, QZ.(2003).Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening.PROTEIN EXPRESSION AND PURIFICATION,27(1),63-74.
MLA	Cheng, DH,et al."Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening".PROTEIN EXPRESSION AND PURIFICATION 27.1(2003):63-74.