Open AccessRecombinant His-tagged DNA polymerase. I. Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase.
Author(s) -
Sławomir Dąbrowski,
Józef Kur
Publication year - 1998
Publication title -
acta biochimica polonica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.452
H-Index - 78
eISSN - 1734-154X
pISSN - 0001-527X
DOI - 10.18388/abp.1998_4258
Subject(s) - thermus thermophilus , dna polymerase , recombinant dna , microbiology and biotechnology , polymerase , thermus aquaticus , dna polymerase i , thermostability , biology , affinity chromatography , molecular cloning , dna polymerase ii , biochemistry , dna clamp , escherichia coli , dna , polymerase chain reaction , enzyme , gene , reverse transcriptase , peptide sequence
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.