Jundishapur Journal of Microbiology (2008); 1(1): 1-5 1 __________________________________________________________________________________________ Cloning and Expression of Thermus aquaticus DNA polymerase in Escherichia coli Mohammad Roayaei, Hamid Galehdari Department of Biology, Faculty of Science, Shahid Chamran University, Ahvaz, Iran Received: April 2008 Accepted: August 2008 Abstract Thermostable DNA polymerase gene from Thermus aquaticus was cloned into constructed Taq from Thermus a Qaticus (pTTQ) plasmid using EcoRI and SalI sites with subsequent transformation in Escherichia coli strain (TOP10). The use of Isopropyl-β-D- thiogalactopyranosid (IPTG) as inducer of interested gene expression under control of the lac promoter was investigated. The optimization of enzyme induction by IPTG was determined at shake flask level to be 0.52mM at exponential growth phase. Enzyme preparation was performed by lysis the cultured cells. Afterwards, the cell suspension was incubated at 75°C to denature all heat sensitive proteins in the cell suspension that have been removed by subsequent centrifugation. Finally, the clarified supernatant containing heat resistant Taq DNA polymerase was collected and stored at -80°C. The activity of enzyme was compared with commercial Taq DNA polymerase, which remained when stored in buffer containing 50% glycerol, at -20°C. The purified enzyme had a molecular weight of 94 KDa, as estimated by SDS-PAGE and yielded appropriate enzyme activity comparing to the commercial Taq DNA polymerase. Keywords: Taq DNA Polymerase, E. coli, expression, Thermus aquaticus Introduction Thermostable DNA polymerase is a very important enzyme for molecular biological studies such as DNA amplification and DNA sequencing by the polymerase chain reaction (PCR) [1, 2]. Most of the thermostable DNA polymerases have been isolated from Thermus aquaticus, a thermostable bacterium, known as Taq polymerase. Taq DNA polymerase is an enzyme obtained from a heat stable bacterium called T. aquaticus having a molecular weight of about 6.6×10 4 –9.4×10 4 Daltons [3]. T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents [4]. Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR [5]. Therefore it replaced the DNA polymerase from E. coli originally used in PCR [6]. Taq's temperature optimum for activity is 75-80°C, with a half-life of 9 minutes at 97.5°C, and can replicate a 10 3 base pair strand of DNA in less than 10 seconds at 72°C [7]. Taq DNA polymerase catalyzes the incorporation of dNTPs into DNA. It requires a DNA template, a primer terminus, and the divalent cation Mg++. Taq polymerase contains a polymerization dependent 5'-3' exonuclease activity. It does not have a 3'-5' exonuclease and thus no