- Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
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Amir Ghasemi, Sobhan Ghafourian, Sedighe Vafaei, Reza Mohebi, Maryam Farzi, Morovat Taherikalani, Nourkhoda Sadeghifard
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Osong Public Health Res Perspect. 2015;6(6):336-340. Published online December 31, 2015
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DOI: https://doi.org/10.1016/j.phrp.2015.10.003
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 Abstract
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- 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.
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Citations
Citations to this article as recorded by  - Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465
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BioMed Research International.2017; 2017: 1. CrossRef
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