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

Donghang Cheng, Qiang Shen, Fajun Nan, Zhen Qian, Qi Zhuang Ye

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)63-74
Number of pages12
JournalProtein Expression and Purification
Volume27
Issue number1
DOIs
StatePublished - Jan 1 2003

Fingerprint

Gelatinases
Fibronectins
Purification
Zinc
Catalytic Domain
Screening
Throughput
Ions
Calcium
Matrix Metalloproteinase 2
Matrix Metalloproteinase Inhibitors
Chromogenics
Dialysis
Substrates
Gelatin
Edetic Acid
Proteolysis
Inhibitory Concentration 50
Buffers
Collagen

Keywords

  • Catalytic domain
  • Fibronectin
  • Gene expression
  • High-throughput screening
  • Human gelatinase A
  • Matrix metalloproteinase

ASJC Scopus subject areas

  • Biotechnology

Cite this

Purification and characterization of catalytic domains of gelatinase a with or without fibronectin insert for high-throughput inhibitor screening. / Cheng, Donghang; Shen, Qiang; Nan, Fajun; Qian, Zhen; Ye, Qi Zhuang.

In: Protein Expression and Purification, Vol. 27, No. 1, 01.01.2003, p. 63-74.

Research output: Contribution to journalArticle

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