Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis
171 amino acids with a molecular weight (MW) of 19.9182 kDa and an isoelectric point of 5.24. The cDNA sequence of S. cucullata ferritin was constructed into a pET-32a expression system for expressing its relative protein efficiently in the Escherichia coli BL21 strain under isopropyl-beta-D-thiogal...
Published in: | Fish & Shellfish Immunology |
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Online Access: | https://doi.org/10.1016/j.fsi.2011.06.020 http://dspace.xmu.edu.cn/handle/2288/11931 |
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ftxiamenuniv:oai:dspace.xmu.edu.cn:2288/11931 2023-05-15T15:59:05+02:00 Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis Zhu, Bo Lin, Qing Ke, Cai-Huan Huang, He-Qing 黄河清 2011-06-28 https://doi.org/10.1016/j.fsi.2011.06.020 http://dspace.xmu.edu.cn/handle/2288/11931 en eng ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD Fish Shellfish Immunol. 2011 Sep;31(3):453-61 1050-4648 http://dx.doi.org/doi:10.1016/j.fsi.2011.06.020 WOS:000294150800012 http://dspace.xmu.edu.cn/handle/2288/11931 Saccostrea cucullata Ferritin Monitoring level of Cd(2+) pollution Bacterial challenge Cisplatin-subunit analysis Bacterial challenge Article 2011 ftxiamenuniv https://doi.org/10.1016/j.fsi.2011.06.020 2020-07-21T11:22:29Z 171 amino acids with a molecular weight (MW) of 19.9182 kDa and an isoelectric point of 5.24. The cDNA sequence of S. cucullata ferritin was constructed into a pET-32a expression system for expressing its relative protein efficiently in the Escherichia coli BL21 strain under isopropyl-beta-D-thiogalactoside (IPTG) induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately MW 20 kDa using both SDS-PAGE and mass spectrometry. S. cucullata ferritin (ScFer) showed 98% identity with Crassostrea gigas ferritin at the amino acid level. The secondary structure and phosphorylation sites of deduced amino acids were predicted with ExPASy proteomics tools and the NetPhos 2.0 server, respectively, and the subunit space structure of recombinant S. cucullata ferritin (rScFer) was built using the molecular operating environmental software system. The results of both in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was ScFer. ICP-MS indicated that rScFer subunit can directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 22.9 CDDP/ferritin subunit for forming a novel complex of CDDP-subunit, which suggests that it constructs a nanometer CDDP core-ferritin for developing a new drug of anti-cancer. The results of both the real-time PCR and Western blotting showed that the expression of ScFer mRNA was up-regulated in the oyster under the stress of Cd(2+). In addition, the expression increment of ScFer mRNA under bacterial challenge indicated that ferritin participated in the immune response of S. cucullata. The recombinant ScFer should prove to be useful for further study of the structure and function of ferritin in S. cucullata. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved. State Natural Science Fund[30870515]; 973 Projects[2010CB126403]; PCSIRT, China[IRT0941] Article in Journal/Newspaper Crassostrea gigas Xiamen University Institutional Repository Fish & Shellfish Immunology 31 3 453 461 |
institution |
Open Polar |
collection |
Xiamen University Institutional Repository |
op_collection_id |
ftxiamenuniv |
language |
English |
topic |
Saccostrea cucullata Ferritin Monitoring level of Cd(2+) pollution Bacterial challenge Cisplatin-subunit analysis Bacterial challenge |
spellingShingle |
Saccostrea cucullata Ferritin Monitoring level of Cd(2+) pollution Bacterial challenge Cisplatin-subunit analysis Bacterial challenge Zhu, Bo Lin, Qing Ke, Cai-Huan Huang, He-Qing 黄河清 Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis |
topic_facet |
Saccostrea cucullata Ferritin Monitoring level of Cd(2+) pollution Bacterial challenge Cisplatin-subunit analysis Bacterial challenge |
description |
171 amino acids with a molecular weight (MW) of 19.9182 kDa and an isoelectric point of 5.24. The cDNA sequence of S. cucullata ferritin was constructed into a pET-32a expression system for expressing its relative protein efficiently in the Escherichia coli BL21 strain under isopropyl-beta-D-thiogalactoside (IPTG) induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately MW 20 kDa using both SDS-PAGE and mass spectrometry. S. cucullata ferritin (ScFer) showed 98% identity with Crassostrea gigas ferritin at the amino acid level. The secondary structure and phosphorylation sites of deduced amino acids were predicted with ExPASy proteomics tools and the NetPhos 2.0 server, respectively, and the subunit space structure of recombinant S. cucullata ferritin (rScFer) was built using the molecular operating environmental software system. The results of both in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was ScFer. ICP-MS indicated that rScFer subunit can directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 22.9 CDDP/ferritin subunit for forming a novel complex of CDDP-subunit, which suggests that it constructs a nanometer CDDP core-ferritin for developing a new drug of anti-cancer. The results of both the real-time PCR and Western blotting showed that the expression of ScFer mRNA was up-regulated in the oyster under the stress of Cd(2+). In addition, the expression increment of ScFer mRNA under bacterial challenge indicated that ferritin participated in the immune response of S. cucullata. The recombinant ScFer should prove to be useful for further study of the structure and function of ferritin in S. cucullata. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved. State Natural Science Fund[30870515]; 973 Projects[2010CB126403]; PCSIRT, China[IRT0941] |
format |
Article in Journal/Newspaper |
author |
Zhu, Bo Lin, Qing Ke, Cai-Huan Huang, He-Qing 黄河清 |
author_facet |
Zhu, Bo Lin, Qing Ke, Cai-Huan Huang, He-Qing 黄河清 |
author_sort |
Zhu, Bo |
title |
Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis |
title_short |
Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis |
title_full |
Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis |
title_fullStr |
Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis |
title_full_unstemmed |
Single subunit type of ferritin from visceral mass of Saccostrea cucullata: Cloning, expression and cisplatin-subunit analysis |
title_sort |
single subunit type of ferritin from visceral mass of saccostrea cucullata: cloning, expression and cisplatin-subunit analysis |
publisher |
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD |
publishDate |
2011 |
url |
https://doi.org/10.1016/j.fsi.2011.06.020 http://dspace.xmu.edu.cn/handle/2288/11931 |
genre |
Crassostrea gigas |
genre_facet |
Crassostrea gigas |
op_relation |
Fish Shellfish Immunol. 2011 Sep;31(3):453-61 1050-4648 http://dx.doi.org/doi:10.1016/j.fsi.2011.06.020 WOS:000294150800012 http://dspace.xmu.edu.cn/handle/2288/11931 |
op_doi |
https://doi.org/10.1016/j.fsi.2011.06.020 |
container_title |
Fish & Shellfish Immunology |
container_volume |
31 |
container_issue |
3 |
container_start_page |
453 |
op_container_end_page |
461 |
_version_ |
1766394872936267776 |