Coexpression of the subunits of T7 DNA polymerase from an artificial operon allows one-step purification of active gp5/Trx complex

Protein Expr Purif. 2006 May;47(1):264-72. doi: 10.1016/j.pep.2005.10.016. Epub 2005 Nov 8.


T7 DNA polymerase expression was performed from an artificial operon by cloning its cofactor, thioredoxin, downstream of a N-terminal 9xHis-tagged T7 gene 5 (gp5). Up to 90% of gp5 was soluble in the presence, but not in the absence of thioredoxin coexpression suggesting that free-form thioredoxin assisted solubilization of gp5. Expression and single-step nickel-agarose affinity purification resulted in recovery of an enzyme that was 97% pure. Copurification of thioredoxin was observed and the estimated molar ratio of thioredoxin to gp5 was 1:1 in the purified DNA polymerase complex. Purified T7 DNA polymerase exhibited full polymerase activity compared to the commercial enzyme and required no exogenous thioredoxin for activity.

MeSH terms

  • Bacteriophage T7 / enzymology*
  • Bacteriophage T7 / genetics
  • DNA-Directed DNA Polymerase / biosynthesis
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / isolation & purification*
  • Escherichia coli / genetics
  • Humans
  • Operon / genetics*
  • Protein Subunits / biosynthesis
  • Protein Subunits / genetics*
  • Protein Subunits / isolation & purification*
  • Thioredoxins / genetics
  • Thioredoxins / isolation & purification*
  • Thioredoxins / metabolism


  • Protein Subunits
  • TXN protein, human
  • Thioredoxins
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase