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Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
- Source :
- Osong Public Health and Research Perspectives
- Publication Year :
- 2015
- Publisher :
- Elsevier BV, 2015.
-
Abstract
- ObjectivesIn an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase.MethodsEscherichia 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.ResultsAmylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system.ConclusionThese results indicate that this expression system was appropriate for the production of thermostable α-amylase.
- Subjects :
- inorganic chemicals
Cloning
amylase
thermophile
biology
Chemistry
Thermophile
Public Health, Environmental and Occupational Health
biology.organism_classification
Molecular biology
Hyperthermophile
law.invention
Pyrococcus woesei
enzymes and coenzymes (carbohydrates)
Infectious Diseases
Biochemistry
law
expression
Recombinant DNA
biology.protein
Original Article
Amylase
Gene
recombinant protein
Subjects
Details
- ISSN :
- 22109099
- Volume :
- 6
- Issue :
- 6
- Database :
- OpenAIRE
- Journal :
- Osong Public Health and Research Perspectives
- Accession number :
- edsair.doi.dedup.....f86237724d6ca8c2df68fbc207e05201
- Full Text :
- https://doi.org/10.1016/j.phrp.2015.10.003