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KMID : 0950020060320010077
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Journal of Health Science & Medical Technology
2006 Volume.32 No. 1 p.77 ~ p.81
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Expression and purification of the DNA polymerase from Thermus aquaticus
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Kim Sung-Wook
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Abstract
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Taq DNA polymerase isolated from Thermus aquaticus has been shown to be highly useful in the polymerase chain reaction (PCR) method of amplifying DNA fragments. I amplified the Taq DNA polymerase gene using the proofreading Pfu polymerase and cloned it into the pKK223-3 vector to produce the expression plasmid pKK-Taq. The protein was highly expressed and accumulated in E. coli cells at 37℃ as soluble protein. The protein was purified by heating to denature E. coli proteins, followed by chromatography on heparin column. At this stage, Taq DNA polymerase was pure enough for most applications. The purified Taq DNA polymerase from E. coli was fully functional as a DNA polymerase when the activity of recombinant Taq DNA polymerase was examined in the PCR reaction. Ten units of Taq DNA polymerase amplified the expected 300 and 500 bp fragments. Taq DNA polymerase at 2.5 - 5 units showed a similar amplification efficiency. The Taq DNA polymerase expressed in E. coli can easily be prepared in large quantities, and it appears to have the same activity as that of commercial source.
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KEYWORD
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Thermus aquaticus, DNA polymerase
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