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KMID : 1120220150060060336
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Osong Public Health and Research Perspectives
2015 Volume.6 No. 6 p.336 ~ p.340
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Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
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Ghasemi Amir
Ghafourian Sobhan
Vafaei Sedighe
Mohebi Reza
Farzi Maryam
Taherikalani Morovat
Sadeghifard Nourkhoda
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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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KEYWORD
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amylase, expression, recombinant protein, thermophile
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