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 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.
['Ghasemi|Amir|A|', 'Ghafourian|Sobhan|S|', 'Vafaei|Sedighe|S|', 'Mohebi|Reza|R|', 'Farzi|Maryam|M|', 'Taherikalani|Morovat|M|', 'Sadeghifard|Nourkhoda|N|']
expression amylase recombinant protein thermophile