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A Journal on Biotechnology and Molecular Biology
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
Impact Factor 0,246
Minerva Biotecnologica 2013 June;25(2):95-100
Determination of yield and activity of recombinant Taq polymerase produced in pTTQ18 Vector
Rani R. 1, Mannan A. 1, 2, Yameen M. A. 1, Mirza B. 1 ✉
1 Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan;
2 Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan
Aim: The Taq DNA polymerase is the most commonly used enzyme in modern biotechnology. Being thermostable and capability to polymerize the DNA, this enzyme has been extensively used in PCR, DNA sequencing etc. To cope with the high demand of Taq, recombinant Taq is being produced. The yield and activity of Taq polymerase is determined by many parameters. These include IPTG concentration, IPTG induction period and IPTG and incubation temperature. The current study was to analyze Taq polymerase produced in pTTQ18 Vector as well as to optimize its expression and purification.
Methods: The E. coli strain DH5α was transformed with pTTQ18 vector carrying the Taq polymerase gene.
Results: Maximum expression of Taq polymerase gene was observed at IPTG concentration of 200 µM with induction period of 12 h. In order to optimize purification, the cellular lysate was best incubated at 75 oC. The activity of purified Taq polymerase was also calculated by comparing it with commercial Taq and was found 1 unit/µl while that of commercial Taq was 5 U/µL.
Conclusion: Overall, results conclude that, at optimum conditions, DH5α transformed with pTTQ18 produce efficient level of Taq polymerase that can be use commercially.