Expression and purification of catalytic domain of human macrophage elastase for high-throughput inhibitor screening

Dong Hang Cheng, Qiang Shen, Jing Qian, Zhen Qian, Qizhuang Ye

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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 the 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.

Original languageEnglish (US)
Pages (from-to)143-151
Number of pages9
JournalActa Pharmacologica Sinica
Volume23
Issue number2
StatePublished - 2002
Externally publishedYes

Fingerprint

Macrophages
Pancreatic Elastase
Purification
Catalytic Domain
Screening
Throughput
High-Throughput Screening Assays
Escherichia coli
Recombinant Proteins
Zinc
Assays
Proteins
Chromogenics
Inclusion Bodies
Chromatography
Biological Products
Anions
Ions
Calcium
Peptides

Keywords

  • Gene expression
  • High-throughput screening
  • Human macrophage elastase
  • Matrix metalloproteinases

ASJC Scopus subject areas

  • Chemistry(all)
  • Pharmacology

Cite this

Expression and purification of catalytic domain of human macrophage elastase for high-throughput inhibitor screening. / Cheng, Dong Hang; Shen, Qiang; Qian, Jing; Qian, Zhen; Ye, Qizhuang.

In: Acta Pharmacologica Sinica, Vol. 23, No. 2, 2002, p. 143-151.

Research output: Contribution to journalArticle

@article{9d81e9c0ab1b4c869a7600de071ff88b,
title = "Expression and purification of catalytic domain of human macrophage elastase for high-throughput inhibitor screening",
abstract = "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 the 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.",
keywords = "Gene expression, High-throughput screening, Human macrophage elastase, Matrix metalloproteinases",
author = "Cheng, {Dong Hang} and Qiang Shen and Jing Qian and Zhen Qian and Qizhuang Ye",
year = "2002",
language = "English (US)",
volume = "23",
pages = "143--151",
journal = "Zhongguo yao li xue bao = Acta pharmacologica Sinica",
issn = "1671-4083",
publisher = "Nature Publishing Group",
number = "2",

}

TY - JOUR

T1 - Expression and purification of catalytic domain of human macrophage elastase for high-throughput inhibitor screening

AU - Cheng, Dong Hang

AU - Shen, Qiang

AU - Qian, Jing

AU - Qian, Zhen

AU - Ye, Qizhuang

PY - 2002

Y1 - 2002

N2 - 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 the 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.

AB - 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 the 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.

KW - Gene expression

KW - High-throughput screening

KW - Human macrophage elastase

KW - Matrix metalloproteinases

UR - http://www.scopus.com/inward/record.url?scp=0036163189&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036163189&partnerID=8YFLogxK

M3 - Article

VL - 23

SP - 143

EP - 151

JO - Zhongguo yao li xue bao = Acta pharmacologica Sinica

JF - Zhongguo yao li xue bao = Acta pharmacologica Sinica

SN - 1671-4083

IS - 2

ER -