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Recombinant His-tagged DNA polymerase. I. Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase

Dabrowski, S.; Kur, J.

Acta Biochimica Polonica 45(3): 653-660

1998

ISSN/ISBN: 0001-527X
PMID: 9918491
Document Number: 486235
The Tth DNA polymerase gene from the thermophilic Thermus thermophilus (strain HB8) was amplified, cloned and expressed in Escherichia coli. The recombinant DNA polymerase containing a polyhistidine tag at the N-terminus was isolated in a single step by Ni2+ affinity chromatography. The purified recombinant enzyme, showing high polymerase activity contained 43 additional amino-acid residues (including a cluster of six histidine residues inserted for purification of the recombinant protein by metal-affinity chromatography) at N-terminus. The applied overexpression system was very efficient giving 700,000 u of DNA polymerase activity from 1 liter of induced culture. The enzyme was characterized and displayed high DNA polymerase and reverse transcriptase activities and high thermostability as compared to the native Tth DNA polymerase.

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