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

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.