Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization

Michiyasu Itoh, Kazuto Masuda, Yuko Ito, Toshifumi Akizawa, Masanori Yoshioka, Kazushi Imai, Yasunori Okada, Hiroshi Sato, Motoharu Seiki

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34 Citations (Scopus)

Abstract

Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors.

Original languageEnglish
Pages (from-to)667-673
Number of pages7
JournalJournal of biochemistry
Volume119
Issue number4
DOIs
Publication statusPublished - 1996 Apr

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Keywords

  • MMP inhibitors
  • Ni-NTA resin
  • Recombinant MMP-7
  • Recombinant matrylisin
  • Zymogen activation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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