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Cloning, Isolation and Characterization of Taq DNA Polymerase Using pET Expression System

Y. Kamal Deepthi and Ramesh M.
Department of Biotechnology and Pharmacy, Jawaharlal Nehru Technological University Kakinada, Vizianagaram, 535003, India.
Abstract—The thermostable properties of the Taq DNA Polymerase (Taq) from Thermus aquaticus ( T. aquaticus ) have contributed greatly to yield, specificity, automation and utility in the Polymerase Chain Reaction (PCR) for amplification of DNA. Isolation of this protein from T. aquaticus is difficult leading to poor yield and quality. Several studies have shown the recombinant expression of Taq DNA Polymerase in heterologous host cells. However, these still suffer from low yields. The use of T7 RNA Polymerase regulated expression vector and availability of affinity tag (His-Tag) can make the expression of Taq DNA Polymerase an efficient and economical process. The project is aimed at recombinant expression of Taq DNA Polymerase in BL 21 host cell line and pET expression vector. Phi4-2 is a Charon 35 (phage) carrying 15 Kb genomic DNA fragment of T. aquaticus genomic DNA and codes for full length Taq DNA Polymerase. Taq DNA Polymerase was amplified from Phi4-2 genomic DNA using a set of primers designed for the amplification of Taq DNA Polymerase gene. Amplified Taq DNA Polymerase was cloned into bacterial expression vector named pET- 24a which has an N-terminal His Tag gene. The vector was then transformed in to BL21 (DE3) competent cells to express Taq. Taq was expressed in the presence of IPTG which is used as an inducer. The expressed protein was purified using Ni-NTA column chromatography method. The activity of the purified protein was then compared with commercial Taq. Inhouse Taq was found to be twice as active as commercial Taq. The enzyme activity of Inhouse Taq could be 2 u/µl.

Index Terms—Taq DNA Polymerase, BL 21 host cell line, Ni-NTA column chromatography

Cite: Y. Kamal Deepthi and Ramesh M., "Cloning, Isolation and Characterization of Taq DNA Polymerase Using pET Expression System," International Journal of Life Sciences Biotechnology and Pharma Research, Vol. 2, No. 1, pp. 267-283, January 2013.
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