Cloning and Expression of Thermus aquaticus DNA polymerase in Escherichia coli

Thermostable DNA polymerase gene from Thermus aquaticus was cloned into constructedTaq from Thermus a Qaticus (pTTQ) plasmid using EcoRI and SalI sites with subsequenttransformation in Escherichia coli strain (TOP10). The use of Isopropyl-β-Dthiogalactopyranosid(IPTG) as inducer of interested gene expression under control of thelac promoter was investigated. The optimization of enzyme induction by IPTG wasdetermined at shake flask level to be 0.52mM at exponential growth phase. Enzymepreparation was performed by lysis the cultured cells. Afterwards, the cell suspension wasincubated at 75°C to denature all heat sensitive proteins in the cell suspension that havebeen removed by subsequent centrifugation. Finally, the clarified supernatant containingheat resistant Taq DNA polymerase was collected and stored at -80°C. The activity ofenzyme was compared with commercial Taq DNA polymerase, which remained whenstored in buffer containing 50% glycerol, at -20°C. The purified enzyme had a molecularweight of 94 KDa, as estimated by SDS-PAGE and yielded appropriate enzyme activitycomparing to the commercial Taq DNA polymerase.