Production and Expression Optimization of Heterologous Serratiopeptidase
Background: Serratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the...
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Tehran University of Medical Sciences
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doaj-fc24a831f3fb4c058049ea1ff4c9b7642021-01-02T15:42:10ZengTehran University of Medical SciencesIranian Journal of Public Health2251-60852251-60932020-05-01495Production and Expression Optimization of Heterologous SerratiopeptidaseMaryam ROUHANI0Vahideh VALIZADEH1Sara MOLASALEHI2Dariush NOROUZIAN3Department of Nano-Biotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, IranDepartment of Nano-Biotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, IranDepartment of Nano-Biotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, IranDepartment of Nano-Biotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran Background: Serratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the cloning and the expression optimization of serratiopeptidase. Methods: The heat-stable serratiopeptidase (5d7w) was selected as the template. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The recombinant protein was expressed in Esherichia coli BL21 and confirmed by SDS-PAGE and Western blot analysis. Different parameters such as expression vector, culture media, post-induction incubation temperature, inducer concentration, and post-induction incubation time were altered to obtain the highest amount of the recombinant protein. Results: Serratiopeptidase was successfully cloned and expressed under optimized conditions in E. coli which confirmed by western blot analysis. The optimal conditions of expression were determined using pQE30 as vector, cultivating the host bacteria in Terrific Broth (TB) medium, at 37º C, induction by IPTG concentration equal to 0.5 mM, and cells were harvested 4 h after induction. Conclusion: As serratiopeptidase is a multi-potent enzyme, the expressed recombinant protein can be considered as a valuable agent for pharmaceutical applications in further studies. https://ijph.tums.ac.ir/index.php/ijph/article/view/15488SerratiopeptidaseCloningExpression optimization |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Maryam ROUHANI Vahideh VALIZADEH Sara MOLASALEHI Dariush NOROUZIAN |
spellingShingle |
Maryam ROUHANI Vahideh VALIZADEH Sara MOLASALEHI Dariush NOROUZIAN Production and Expression Optimization of Heterologous Serratiopeptidase Iranian Journal of Public Health Serratiopeptidase Cloning Expression optimization |
author_facet |
Maryam ROUHANI Vahideh VALIZADEH Sara MOLASALEHI Dariush NOROUZIAN |
author_sort |
Maryam ROUHANI |
title |
Production and Expression Optimization of Heterologous Serratiopeptidase |
title_short |
Production and Expression Optimization of Heterologous Serratiopeptidase |
title_full |
Production and Expression Optimization of Heterologous Serratiopeptidase |
title_fullStr |
Production and Expression Optimization of Heterologous Serratiopeptidase |
title_full_unstemmed |
Production and Expression Optimization of Heterologous Serratiopeptidase |
title_sort |
production and expression optimization of heterologous serratiopeptidase |
publisher |
Tehran University of Medical Sciences |
series |
Iranian Journal of Public Health |
issn |
2251-6085 2251-6093 |
publishDate |
2020-05-01 |
description |
Background: Serratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the cloning and the expression optimization of serratiopeptidase.
Methods: The heat-stable serratiopeptidase (5d7w) was selected as the template. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The recombinant protein was expressed in Esherichia coli BL21 and confirmed by SDS-PAGE and Western blot analysis. Different parameters such as expression vector, culture media, post-induction incubation temperature, inducer concentration, and post-induction incubation time were altered to obtain the highest amount of the recombinant protein.
Results: Serratiopeptidase was successfully cloned and expressed under optimized conditions in E. coli which confirmed by western blot analysis. The optimal conditions of expression were determined using pQE30 as vector, cultivating the host bacteria in Terrific Broth (TB) medium, at 37º C, induction by IPTG concentration equal to 0.5 mM, and cells were harvested 4 h after induction.
Conclusion: As serratiopeptidase is a multi-potent enzyme, the expressed recombinant protein can be considered as a valuable agent for pharmaceutical applications in further studies.
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topic |
Serratiopeptidase Cloning Expression optimization |
url |
https://ijph.tums.ac.ir/index.php/ijph/article/view/15488 |
work_keys_str_mv |
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