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Original Article
- Cloning, Expression, and Purification of Hyperthermophile α-Amylase from
Pyrococcus woesei - Amir Ghasemi, Sobhan Ghafourian, Sedighe Vafaei, Reza Mohebi, Maryam Farzi, Morovat Taherikalani, Nourkhoda Sadeghifard
- Osong Public Health Res Perspect. 2015;6(6):336-340. Published online December 31, 2015
- DOI: https://doi.org/10.1016/j.phrp.2015.10.003
- 3,682 View
- 28 Download
- 3 Crossref
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Abstract
PDF - 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. -
Citations
Citations to this article as recorded by
- Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465
A. Al-Amri, M. A. Al-Ghamdi, J. A. Khan, H. N. Altayeb, H. Alsulami, M. Sajjad, O. A. Baothman, M. S. Nadeem
Brazilian Journal of Biology.2022;[Epub] CrossRef - Glycoside Hydrolases and Glycosyltransferases from Hyperthermophilic Archaea: Insights on Their Characteristics and Applications in Biotechnology
Khadija Amin, Sylvain Tranchimand, Thierry Benvegnu, Ziad Abdel-Razzak, Hala Chamieh
Biomolecules.2021; 11(11): 1557. CrossRef - Optimization, Purification, and Starch Stain Wash Application of Two Newα-Amylases Extracted from Leaves and Stems ofPergularia tomentosa
Imen Lahmar, Hanen El Abed, Bassem Khemakhem, Hafedh Belghith, Ferjani Ben Abdallah, Karima Belghith
BioMed Research International.2017; 2017: 1. CrossRef
- Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465
Article
- Non-chromatographic Method for the Hepatitis B Virus X Protein Using Elastin-Like Polypeptide Fusion Protein
- Soon-Hwan Kwon, Hyeseong Cho
- Osong Public Health Res Perspect. 2012;3(2):79-84. Published online June 30, 2012
- DOI: https://doi.org/10.1016/j.phrp.2012.04.003
- 3,916 View
- 18 Download
- 5 Crossref
-
Abstract
PDF
- Objectives
Hepatitis B virus (HBV) is a member of the hepadnavirus family. The HBV genome contains four genes designated as S, C, P, and X. The HBV X (HBx) gene encodes for a 16.5-kDa regulatory protein that enhances HBV replication and exerts multifunctional activities. The aim of this study is to describe the rapid and easy purification of HBx using ELP (elastin-like polypeptide) fusion protein.
Methods
The ELP–HBx fusion protein was overexpressed in Escherichia coli. Environmental sensitivity was demonstrated via turbidity and dynamic light scattering as a function of temperature. HBx was purified as an ELP fusion protein. ELPs are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. ELP follows in temperature and salt consistency, precipitation, and solution repetition (inverse transition cycling) with polypeptide, where it purifies the protein in a simple manner.
Results
Fusion proteins underwent supramolecular aggregation at 40 ℃ in 1 M NaCl and slowly resolubilized at subphysiologic temperatures. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity to the commercial HBx.
Conclusion
This study suggests that HBx can be purified rapidly and easily using inverse transition cycling, and that this method can be applied in determination of HBx 3D structures and HBx stability study. -
Citations
Citations to this article as recorded by- Evaluation of machine learning algorithms to predict the hydrodynamic radii and transition temperatures of chemo-biologically synthesized copolymers
Jared S. Cobb, Maria A. Seale, Amol V. Janorkar
Computers in Biology and Medicine.2021; 128: 104134. CrossRef - Machine learning to determine optimal conditions for controlling the size of elastin-based particles
Jared S. Cobb, Alexandra Engel, Maria A. Seale, Amol V. Janorkar
Scientific Reports.2021;[Epub] CrossRef - Influence of miR‐520e‐mediated MAPK signalling pathway on HBV replication and regulation of hepatocellular carcinoma cells via targeting EphA2
Jing‐hui Tian, Wen‐dong Liu, Zhi‐yong Zhang, Li‐hua Tang, Dong Li, Zhao‐ju Tian, Shao‐wei Lin, Ying‐jie Li
Journal of Viral Hepatitis.2019; 26(4): 496. CrossRef - Elastin‐like polypeptides: A strategic fusion partner for biologics
Agnes Yeboah, Rick I. Cohen, Charles Rabolli, Martin L. Yarmush, Francois Berthiaume
Biotechnology and Bioengineering.2016; 113(8): 1617. CrossRef - Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers
Tomasz Kowalczyk, Katarzyna Hnatuszko-Konka, Aneta Gerszberg, Andrzej K. Kononowicz
World Journal of Microbiology and Biotechnology.2014; 30(8): 2141. CrossRef
- Evaluation of machine learning algorithms to predict the hydrodynamic radii and transition temperatures of chemo-biologically synthesized copolymers
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