Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

Plasmid. 2008 May;59(3):231-7. doi: 10.1016/j.plasmid.2008.01.001. Epub 2008 Mar 4.


We describe the construction and use of two sets of vectors for the over-expression and purification of protein from Escherichia coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His(6)) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His(6) and maltose-binding protein tag in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / chemistry
  • Citrates / chemistry
  • Cloning, Molecular
  • Endopeptidases / metabolism
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Genetic Techniques*
  • Genetic Vectors*
  • Histidine / chemistry
  • Hydro-Lyases / genetics
  • Models, Genetic
  • Oligopeptides / chemistry
  • Plasmids / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Proteins / chemistry
  • Salmonella enterica / enzymology
  • Shewanella / metabolism


  • Bacterial Proteins
  • Citrates
  • Escherichia coli Proteins
  • His-His-His-His-His-His
  • Oligopeptides
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Histidine
  • 2-methylcitric acid
  • Endopeptidases
  • TEV protease
  • Hydro-Lyases
  • methylcitrate dehydratase, E coli