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KMID : 0578319910010030369
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Molecules and Cells
1991 Volume.1 No. 3 p.369 ~ p.375
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Enhanced Expression I Escherichia coli of Cloned Thermus Aquaticus DNA Polymerase Gene by Optimized Distance between shine-Dalgarno Sequence and ATG Codon
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Kwon, Wuk-Tae
Kim, Joong Su/Park, Jong Hoon/Koh, Sukhoon/Lee, Dae-Sil
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Abstract
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Thermus aquaticus YT-1 (Taq) DNA polymerase has been known to be highly useful for amplifying DNA fragments by polymerase chain reaction (PCR), and for DNA sequencing. This study covers the cloning and expression of Taq DNA polymerase gene in Escherichia coli, and the purification of the enzyme. The DNA fragments encoding DNA polymerase from T. aquaticus YT-1 were cloned into E. coli using both PCR method and hybridization technique. A series of expression vector pKTPOL´s for Taq DNA polymerase gene were constructed and then expressed in E. coli. The expression level from E. coli harboring plasmid pKTPOLI7 was initially observed to be rather low. Optimal expression levels of Taq DNA polymerase gene have been obtained by adjusting the distance between the Shine-Dalgarno sequence and the ATG start codon. Taq DNA polymerase in E. coli was purified by simple methods like heat treating. The purified enzyme was shown to have equal activity, especially for DNA amplification by PCR, compared with that of authentic enzymes.
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