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

Acta Biochim Pol. 1998;45(3):653-60.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cloning, Molecular
  • DNA, Bacterial
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / isolation & purification
  • Electrophoresis, Polyacrylamide Gel
  • Histidine / chemistry*
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Thermus thermophilus / enzymology*

Substances

  • DNA, Bacterial
  • Recombinant Proteins
  • Histidine
  • DNA polymerase, Thermus thermophilus
  • DNA-Directed DNA Polymerase