Expression and purification of histidine-tagged bacteriophage T7 DNA polymerase

Protein Expr Purif. 2005 Feb;39(2):247-53. doi: 10.1016/j.pep.2004.10.022.


The formation of inclusion bodies is a frequent consequence of high-level production of foreign protein in the cytoplasm of Escherichia coli. This phenomenon is also observed with bacteriophage T7 gene 5 protein, the phage-encoded subunit of T7 DNA polymerase, if expression is based on the T5 promoter/lac operator transcription-translation system present in a vector with ColE1 origin of replication. To avoid tedious procedures for recovering protein from insoluble aggregates, we studied the expression of T7 gene 5 protein using a series of E. coli strains, and optimized the yield of soluble, histidine-tagged (His-tagged) protein by varying the respective growth conditions (temperature, amount of inducer isopropyl-beta-d-thiogalactopyranoside, and presence of organic osmolytes). Although the expression levels in three different strains (BL21, SG13009, and XL1-Blue) were almost comparable with a given set of growth conditions, the yields of soluble protein differed markedly. The largest quantities of soluble, His-tagged T7 gene 5 protein were achieved using "cloning strain" XL1-Blue which benefitted significantly from the presence of sorbitol and glycine betaine-in contrast to the expression strains BL21 and SG13009. Purification of His-tagged T7 gene 5 protein was achieved using single-step metal-affinity chromatography that yielded large amounts of highly active polymerase (97% homogeneity). The application of this expression/purification approach represents not only a useful method to purify large quantities of T7 DNA polymerase for structural investigations but also, provides a fast and efficient protocol for the parallel purification of T7 DNA polymerase variants, e.g., for automated screenings or directed evolution experiments.

Publication types

  • Comparative Study

MeSH terms

  • Bacteriophage T7 / enzymology
  • Bacteriophage T7 / genetics*
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / isolation & purification*
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Escherichia coli / metabolism
  • Fluorometry
  • Gene Expression*
  • Genetic Vectors
  • Histidine / chemistry
  • Kinetics
  • Plasmids
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Transformation, Genetic


  • Recombinant Proteins
  • Histidine
  • DNA-Directed DNA Polymerase