EXPRESSION AND PURIFICATION OF DNA POLYMERASE FROM THERMUS THERMOPHILUSINE.COLI EXPRESSION SYSTEM

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Authors

P. Li

National Center for Biotechnology, 13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan

S. Abeldenov

National Center for Biotechnology, 13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan

B. Khassenov

National Center for Biotechnology, 13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan

Abstract

Isolation of thermostable DNA polymerase from Thermusaquaticus was a significant stage in molecular biology and laboratory performance. After Taq DNA polymerase was discovered PCR method became widespread among laboratories around the world. Taq DNA polymerase was the first tool for fast and highly specific amplification of targeted nucleotide sequences. Thus, thermostable polymerases became vital for laboratory performance.

We managed to express and purify recombinant Tth DNA polymerase that has both polymerase and reverse transcriptase activities. At first we cultivated Thermusthermophilus strain HB8 and isolated genomic DNA. The gene was amplified and cloned into expression plasmid vector pET-28c(+) under T7 promoter. E.coli cells BL-21(DE3) were transformed with obtained recombinant vector and cultivated with kanamycin antibiotic in LB-broth. Induction was started with IPTG. Cells were disrupted by lysozyme and ultrasonication. Liquid fraction was loaded into sepharose column.

Obtained purified enzyme is highly thermostable which was tested in a condition of high temperature and are able to preserve polymerase activity even after heating at 95℃ during 30 minutes. Recombinant Tth polymerase has 95% SDS-PAGE purity. Also we have managed to made different storage and reaction buffers (with various concentrations of salts, stabilizers and detergents) in order to determine the best combination.

Keywords

Tth, Thermusthermophilus, recombinant protein, E.coli, polymerase, enzyme

Article Details

References

Tabor S., Richardson C.C. DNA sequence analisys with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA, 1987,vol. 84, рр. 467-4771.

Innis M.A., Gelfand D.H., Sninsky J.J., White T.J PCR protocols: A guide to methods and applications.Academic Press, 1990, рр. 146-152.

Klimczak L.J., Grummt F., Burger K.J. Purification and characterization of DNA polymerase from archaebacteriumSulfolobusacidocaldarius.Nucleic acid research, 1985,vol.13,рр. 5269-5282.

Rella R., Raia C.A., Pisani F.M., D'Auria S., Nucci R., Gambacorta A., de Rosa M., Rossi M. Purification and properties of themophilic thermostable DNA polymerase from the archaebacteriumSulfolobussolfataricus.Italial Journal of Biochemistry, 1990,vol. 39, рр. 83-99.

Elie C., de Recondo A.M., Forterre P. Thermostable DNA polymerase from the archaebacteriumSulfolobusacidocaldarius. Purification, characterization and immunological properties.European journal of biochemistry, 1989,vol. l, no.17, pp. 619-626.

Oshima&Imahori, K. Description of Thermusthermophilus (Yoshida and Oshima) comb. nov., a nonsporulating thermophilic bacterium from Japanese thermal spa.Int J SystBacteriol., 1974,vol. 24, pp. 102-112.

Molecular cloning. A laboratory manual / T. Maniatis, J. Sambrook. New York, Cold Spring Harbor Laboratory, 1982, pp. 545.

Abeldenov S.,KhassenovB. Cloning, expression and purification of recombinant analog of Taq DNA polymerase.Biotechnology. Theory and Practice, 2014,no. 1, pp. 12-16.

Mussakhmetov A.N.A., Abeldenov S., Khassenov B. Purification of recombinant Pfu DNA polymerase by double step affinity chromatography.Biotechnology. Theory and Practice, 2014, no. 2, pp. 42-47.

Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem., 1976,vol. 72, pp. 248-254.

Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature, 1970,vol. 227, no. 5259, pp. 680-685.