Cloning, Expression and Purification of Hyperthermophile α-amylase from

An α-amylase gene recognized in the reported genome sequence of Pyrococcus woesei was amplified and cloned into pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. Escherichia coli BL-21 (DE3) was transformed with this plasmid and the enzyme was expressed over induction with IPTG. The expressed protein appeared at a position corresponding to ∼100 kDa on SDS-PAGE with intein tag. Amylolytic activity of proteins produced by transformed E. coli cells was detected by Zymogram and the rate of active α-amylase with and without intein in both soluble condition and inclusion body solubilized by 4M urea solution were measured.
Source: Osong Public Health and Research Perspectives - Category: Global & Universal Authors: Tags: Original Article Source Type: research