Osong Public Health Res Perspect.  2015 Dec;6(6):336-340. 10.1016/j.phrp.2015.10.003.

Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

• Ghasemi A
• Ghafourian S
• Vafaei S
• Mohebi R
• Farzi M
• Taherikalani M
• Sadeghifard N

• KMID: 2518227
• DOI: http://doi.org/10.1016/j.phrp.2015.10.003

Abstract

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.

Keyword

amylase; expression; recombinant protein; thermophile