caa a22 4500 397557566 CHVBK 20180308164807.0 cr unu---uuuuu 161202e199604 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021294 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021294 Purification and Refolding of Recombinant Human proMMP-7 (pro-Matrilysin) Expressed inEscherichia coli and Its Characterization [Elektronische Daten] [Michiyasu Itoh, Kazuto Masuda, Yuko Ito, Toshifumi Akizawa, Masanori Yoshioka, Kazushi Imai, Yasunori Okada, Hiroshi Sato, Motoharu Seiki] 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. © by the Journal of Biochemistry Regular Papers nationallicence Ni-NTA resin nationallicence MMP inhibitors nationallicence recombinant matrylisin nationallicence recombinant MMP-7 nationallicence zymogen activation nationallicence Itoh Michiyasu Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut Masuda Kazuto Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut Ito Yuko Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut Akizawa Toshifumi Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut Yoshioka Masanori Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut Imai Kazushi Departments of Molecular Immunology and Pathology Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut Okada Yasunori Departments of Molecular Immunology and Pathology Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut Sato Hiroshi Departments of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut Seiki Motoharu Departments of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut The Journal of Biochemistry Oxford University Press 119/4(1996-04), 667-673 0021-924X 119:4<667 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021294 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021294 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Itoh Michiyasu Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut NATIONALLICENCE 700 1- Masuda Kazuto Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut NATIONALLICENCE 700 1- Ito Yuko Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut NATIONALLICENCE 700 1- Akizawa Toshifumi Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut NATIONALLICENCE 700 1- Yoshioka Masanori Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 aut NATIONALLICENCE 700 1- Imai Kazushi Departments of Molecular Immunology and Pathology Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 700 1- Okada Yasunori Departments of Molecular Immunology and Pathology Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 700 1- Sato Hiroshi Departments of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 700 1- Seiki Motoharu Departments of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University 13-1 Takara-machi, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/4(1996-04), 667-673 0021-924X 119:4<667 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford