Advanced Biomedical Research
Optimization of Taq DNA polymerase enzyme expression in Escherichia coli
Authors
- Fateme Moazen 1
- Ali Rastegari 1
- Sayed Mehdi Hoseini 1
- Mojtaba Panjehpour 2
- Mehran Miroliaei 3
- Hamid Mir Mohammad Sadeghi 1
1 Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Science, Isfahan, Iran
2 Department of Clinical Biochemistry, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Science, Isfahan, Iran
3 Department of Biology, School of Basic Sciences, University of Isfahan, Isfahan, Iran
Abstract
Background: In the present study, we optimized the experimental conditions using pET15b expression vector to obtain large amounts of Taq DNA polymerase.
Materials and Methods: Correct framing of the gene in the expression vector pET15b and its orientation were analyzed by digestion and sequencing. Production of Taq DNA polymerase in Escherichia coli BL21 (DE3) cells was induced by incubation with different concentrations of IPTG. Optimum production occurred with the addition of 1mM IPTG for 2h. The activity of the obtained enzyme was measured by comparing the intensities of the produced DNA bands in PCR reactions.
Results: Recombinant plasmid containing taq polymerase gene was confirmed by restriction digestion and DNA sequencing. Purified protein was identified by Western blotting. Optimum condition for the production of the enzyme was induction with 1mM IPTG for 23h. Addition of NP40 increased enzyme stability.
Conclusion: We expressed the recombinant Taq DNA polymerase in E. coli using a T7based promoter system and obtained an active and stable enzyme.
Keywords
| 1. | McPherson MJ and Moller SG. PCR. UK: BIOS Scientific Publishers Limited; 2000. p. 35.  |
| 2. | Manzur MJ, Munoz RV, Lucero AA, Ayub MJ, Alvares SE, Ciuffo GM. Production of recombinant enzymes of wide use for Research. Electron J Biotechnol 2006;9:291-3. |
| 3. | Lawyer F, Stoffel S, Saiki R, Chang S, Landre P, Abramson R. High level expression , purification, and enzymatic characterization of full length thermus aquaticus DNA polymerase and a truncated form deficient in 5´ to 3´ exonuclease activity. PCR Methods Appl 1993;2:275-87. |
| 4. | Desai UJ, Pfaffle PK. Single-step purification of thermostable DNA polymerase expressed in Escherichia coli. Biotechniques 1995;19:780-4. |
| 5. | Engelke DR, Krikos A, Bruck ME, Ginsburg D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal Biochem 1990;191:396-400. |
| 6. | Pluthero FG. Rapid purification of high activity Taq DNA polymerase. Nucleic Acids Res 1993;21:4850-1. |
| 7. | Sadeghi HM, Rabbani M, Moazen F. Amplification and cloning of Taq DNA polymerase gene from Thermus Aquaticus strain YT-1. Res Pharm Sci 2006 ; 1:49-52. |
| 8. | Dormiani K, Khazaei Y, Sadeghi HM, Rabbani M, Moazen F. Cloning and expression of a human tissue plasminogen activator variant: K2S in Echerichia coli. Pak J Biol Sci 2007;10:946-9. |
| 9. | Sambrook J. Fritsch EF and Maniatis T. Molecular cloning: A laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory; 2001. p. A843-54. |
| 10. | Sadeghi HM, Rabbani M, Moazen F, Homami S. Molecular detection of lipase gene in a putative Bacillus subtilis strain isolated from soil. Iran. J. Biotechnol 2010;8:49. |
| 11. | Grim E and Arbuthnot P. Rapid purification of recombinant Taq DNA polymerase by freezing and high temperature thawing of bacterial expression cultures. Nucleic Acids Res 1995;23:4518-9. |
| 12. | Baneyx F. Recombinant protein expression in Escherichia Coli. Curr Opin Biotechnol 1990;10:411-21. |
| 13. | Schumann W and Ferreira LC. Production of recombinant proteins in Escherichia Coli. Genet Mol Biol 2004;27:442-53. |
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