Asian Journal of Research in Biochemistry, XXXX,Vol.: 2, Issue.: 4
High-level Expression and Purification of DNA and DNase Free Taq DNA Polymerase
Mukti Nath Mishra1, Jeevanandam Mohanraj1, S. Durairaju Nisshanthini1 and Shama Bhat1* 1Bhat Biotech India Pvt. Ltd., Bangalore, Karnataka, India.
Mukti Nath Mishra1, Jeevanandam Mohanraj1, S. Durairaju Nisshanthini1 and Shama Bhat1*
1Bhat Biotech India Pvt. Ltd., Bangalore, Karnataka, India.
(1) Dr. Mohamed Atiega Elbagermi, Associate Professor, Department of Chemistry, Misurata University, Libya.
(1) Sabina Kędzierska-Mieszkowska, University of Gdańsk, Poland.
(2) Jin Seop Bak, Kyonggi University, Korea.
(3) Keith Stine, University of Missouri, USA.
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Aim: Taq DNA polymerase from Thermus aquaticus is a key enzyme in the field of molecular biology that has been mostly used in polymerase chain reaction (PCR). Our aim is to produce standard grade Taq DNA polymerase free from censorious impurities like DNase, DNA and other contaminating proteins.
Place and Duration of Study: The experiments were performed at the Molecular Diagnostic Division of Bhat Biotech India (P) Ltd., Bangalore from February 2017 to January 2018.
Methodology: The recombinant Taq DNA polymerase clone was confirmed by PCR and DNA sequencing followed by BLAST analysis. The recombinant protein was in soluble form and was expressed by E. coli DH5α strain. The enzyme was extracted using the boil-lysis method and followed by purification with ion exchange chromatography and silica column chromatography to remove the contaminating protein, DNase and DNA. The yield of the protein was also calculated.
Results: In our laboratory, high-quality Taq DNA polymerase was purified using ion exchange chromatography columns and silica column, with a resulting yield of about 45-50 mg/L and the activity was found to be 1.5 U/µL.
Conclusion: The use of a silica column to remove the residual DNA is a remarkable step in obtaining an unequalled quality of Taq DNA polymerase.
Taq DNA polymerase; Thermus aquaticus; DNase; PCR; BLAST; chromatography.
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