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Maryam Farzi 1 Article
Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
Amir Ghasemi, Sobhan Ghafourian, Sedighe Vafaei, Reza Mohebi, Maryam Farzi, Morovat Taherikalani, Nourkhoda Sadeghifard
Osong Public Health Res Perspect. 2015;6(6):336-340.   Published online December 31, 2015
DOI: https://doi.org/10.1016/j.phrp.2015.10.003
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AbstractAbstract PDF
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.

PHRP : Osong Public Health and Research Perspectives