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Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
OBJECTIVES: In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. METHODS: Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protei...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Korea Centers for Disease Control and Prevention
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700765/ https://www.ncbi.nlm.nih.gov/pubmed/26835242 http://dx.doi.org/10.1016/j.phrp.2015.10.003 |
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author | Ghasemi, Amir Ghafourian, Sobhan Vafaei, Sedighe Mohebi, Reza Farzi, Maryam Taherikalani, Morovat Sadeghifard, Nourkhoda |
author_facet | Ghasemi, Amir Ghafourian, Sobhan Vafaei, Sedighe Mohebi, Reza Farzi, Maryam Taherikalani, Morovat Sadeghifard, Nourkhoda |
author_sort | Ghasemi, Amir |
collection | PubMed |
description | OBJECTIVES: In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. METHODS: Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured. RESULTS: Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system. CONCLUSION: These results indicate that this expression system was appropriate for the production of thermostable α-amylase. |
format | Online Article Text |
id | pubmed-4700765 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Korea Centers for Disease Control and Prevention |
record_format | MEDLINE/PubMed |
spelling | pubmed-47007652016-02-01 Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei Ghasemi, Amir Ghafourian, Sobhan Vafaei, Sedighe Mohebi, Reza Farzi, Maryam Taherikalani, Morovat Sadeghifard, Nourkhoda Osong Public Health Res Perspect Original Article OBJECTIVES: In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. METHODS: Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured. RESULTS: Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system. CONCLUSION: These results indicate that this expression system was appropriate for the production of thermostable α-amylase. Korea Centers for Disease Control and Prevention 2015-12 2015-10-20 /pmc/articles/PMC4700765/ /pubmed/26835242 http://dx.doi.org/10.1016/j.phrp.2015.10.003 Text en Copyright © 2015 Korea Centers for Disease Control and Prevention. Published by Elsevier Korea LLC. All rights reserved. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Original Article Ghasemi, Amir Ghafourian, Sobhan Vafaei, Sedighe Mohebi, Reza Farzi, Maryam Taherikalani, Morovat Sadeghifard, Nourkhoda Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei |
title | Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei |
title_full | Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei |
title_fullStr | Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei |
title_full_unstemmed | Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei |
title_short | Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei |
title_sort | cloning, expression, and purification of hyperthermophile α-amylase from pyrococcus woesei |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700765/ https://www.ncbi.nlm.nih.gov/pubmed/26835242 http://dx.doi.org/10.1016/j.phrp.2015.10.003 |
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