Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
10.1016/j.phrp.2015.10.003
- Author:
Amir GHASEMI
1
;
Sobhan GHAFOURIAN
;
Sedighe VAFAEI
;
Reza MOHEBI
;
Maryam FARZI
;
Morovat TAHERIKALANI
;
Nourkhoda SADEGHIFARD
Author Information
1. Department of Microbiology and Immunology, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran
- Publication Type:Original Article
- From:
Osong Public Health and Research Perspectives
2015;6(6):336-340
- CountryRepublic of Korea
- Language:English
-
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.
