Objectives
In an attempt α-amylase gene from <i>Pyrococcus woesei</i> was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. Methods
<i>Escherichia coli</i> 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 <i>E. coli</i> 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
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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 <i>Escherichia coli</i>. 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.
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