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Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing

BACKGROUND: Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia Sericata (L. sericata) collagenase (MMP-...

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Autores principales: Alipour, Hamzeh, Raz, Abbasali, Dinparast Djadid, Navid, Zakeri, Sedigheh
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Institute of Genetic Engineering and Biotechnology 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357693/
https://www.ncbi.nlm.nih.gov/pubmed/32671126
http://dx.doi.org/10.30498/IJB.2019.92707
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author Alipour, Hamzeh
Raz, Abbasali
Dinparast Djadid, Navid
Zakeri, Sedigheh
author_facet Alipour, Hamzeh
Raz, Abbasali
Dinparast Djadid, Navid
Zakeri, Sedigheh
author_sort Alipour, Hamzeh
collection PubMed
description BACKGROUND: Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia Sericata (L. sericata) collagenase (MMP-1) has been expressed in insect cells. Collagenase is an enzyme that is widely used in clinical therapy and industry. It has been indicated that collagenase is expressed and secreted in salivary glands of L. sericata while using for maggot debridement therapy. OBJECTIVES: In the present study we decided to produce the recombinant form of collagenase enzyme in Spodoptera frugiperda (SF9) insect cells using the baculovirus expression system (Bac-to-Bac). MATERIALS AND METHODS: cloned the coding sequences (residues 494-1705) of L. sericata collagenase into the pFastBacHTA as donor plasmid. After transposition in the bacmid of DH10Bac host, the bacmid was transfected into the Sf9 cell line, then the expressed recombinant collagenase (MMP-1) was purified using the Ni-NTA agarose. RESULTS: The recombinant protein was verified by Western blotting. Furthermore, the biological activity of purified protein was measured in the presence of its specific substrate and its inhibitor, which was 67 IU.mL(-1) based on our results, it was revealed that the characterized gene in our previous study codes L. sericata collagenesa enzyme. CONCLUSION: Considering to the broad applications of collagenase in medical sciences, for the first time, we cloned the L. sericata collagenase (MMP-1) gene into the insect cell line to establish a method for the expression and purification of L. sericata collagenase (MMP-1). The result help for preparing and designing a safe and versatile recombinant drug in future.
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spelling pubmed-73576932020-07-14 Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing Alipour, Hamzeh Raz, Abbasali Dinparast Djadid, Navid Zakeri, Sedigheh Iran J Biotechnol Research Article BACKGROUND: Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia Sericata (L. sericata) collagenase (MMP-1) has been expressed in insect cells. Collagenase is an enzyme that is widely used in clinical therapy and industry. It has been indicated that collagenase is expressed and secreted in salivary glands of L. sericata while using for maggot debridement therapy. OBJECTIVES: In the present study we decided to produce the recombinant form of collagenase enzyme in Spodoptera frugiperda (SF9) insect cells using the baculovirus expression system (Bac-to-Bac). MATERIALS AND METHODS: cloned the coding sequences (residues 494-1705) of L. sericata collagenase into the pFastBacHTA as donor plasmid. After transposition in the bacmid of DH10Bac host, the bacmid was transfected into the Sf9 cell line, then the expressed recombinant collagenase (MMP-1) was purified using the Ni-NTA agarose. RESULTS: The recombinant protein was verified by Western blotting. Furthermore, the biological activity of purified protein was measured in the presence of its specific substrate and its inhibitor, which was 67 IU.mL(-1) based on our results, it was revealed that the characterized gene in our previous study codes L. sericata collagenesa enzyme. CONCLUSION: Considering to the broad applications of collagenase in medical sciences, for the first time, we cloned the L. sericata collagenase (MMP-1) gene into the insect cell line to establish a method for the expression and purification of L. sericata collagenase (MMP-1). The result help for preparing and designing a safe and versatile recombinant drug in future. National Institute of Genetic Engineering and Biotechnology 2019-12-01 /pmc/articles/PMC7357693/ /pubmed/32671126 http://dx.doi.org/10.30498/IJB.2019.92707 Text en Copyright: © 2019 The Author(s); Published by Iranian Journal of Biotechnology http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 Unported License, ( http://creativecommons.org/licenses/by-nc/4.0/ ) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Alipour, Hamzeh
Raz, Abbasali
Dinparast Djadid, Navid
Zakeri, Sedigheh
Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing
title Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing
title_full Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing
title_fullStr Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing
title_full_unstemmed Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing
title_short Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing
title_sort expression of a new recombinant collagenase protein of lucilia sericata in sf9 insect cell as a potential method for wound healing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357693/
https://www.ncbi.nlm.nih.gov/pubmed/32671126
http://dx.doi.org/10.30498/IJB.2019.92707
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