Cloning and overexpression of thermostable DNA polymerase gene in Escherichia coli

Chin J Biotechnol. 1995;11(3):185-91.

Abstract

Thermostable DNA polymerase genes had been amplified from Thermus aquaticus YT-1 using the PCR technique. The amplified 2.5-kb DNA fragment was inserted into pUC18 and confirmed to be a thermostable DNA polymerase gene by restriction mapping and DNA sequencing. The insert fragment was then combined into an expression vector, pBV221. This recombinant plasmid overexpressed a 94-kDa of recombinant protein in E. coli. A 100-mL E. coli. culture could yield 1.5 x 10(5) units of Taq DNA polymerase, which could be applied in the PCR.

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • DNA, Bacterial / genetics
  • DNA-Directed DNA Polymerase / genetics*
  • Electrophoresis, Agar Gel
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Gene Expression Regulation, Bacterial / genetics
  • Molecular Sequence Data
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • RNA-Directed DNA Polymerase / genetics
  • Taq Polymerase
  • Temperature

Substances

  • DNA, Bacterial
  • Taq Polymerase
  • RNA-Directed DNA Polymerase
  • DNA-Directed DNA Polymerase