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

Ghasemi, A. and Ghafourian, S. and Vafaei, S. and Mohebi, R. and Farzi, M. and Taherikalani, M. and Sadeghifard, N. (2015) Cloning, Expression, and Purification of Hyperthermophile Amylase from Pyrococcus woesei. Osong Public Health and Research Perspectives, 6 (6). pp. 336-340.

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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. © 2015.

Item Type: Article
Additional Information: cited By 0
Uncontrolled Keywords: amylase; recombinant enzyme, alpha amylase gene; Article; bacterial gene; bacterium culture; cell inclusion; controlled study; enzyme activity; enzyme purification; Escherichia coli; molecular cloning; molecular weight; nonhuman; polyacrylamide gel electrophoresis; priority journal; protein expression; Pyrococcus furiosus; recombinant plasmid; solubility; zymography
Subjects: Immunology and Microbiology
Divisions: Faculty of Medicine > Basic Sciences > Department of Microbiology & Immunology
Depositing User: editor . 2
Date Deposited: 08 Mar 2017 14:56
Last Modified: 08 Mar 2017 14:56
URI: http://eprints.kaums.ac.ir/id/eprint/244

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