Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants

Protein Expr Purif. 2013 Nov;92(1):54-61. doi: 10.1016/j.pep.2013.08.021. Epub 2013 Sep 6.


Short peptide affinity tags have become indispensable in protein research. They cannot only be used for affinity purification but also downstream for detection and assay of an arbitrary fused recombinant protein without the need for any prior knowledge of its biochemical properties. Strep-tag®II is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The affinity receptor for Strep-tag®II is affinity engineered streptavidin, named Strep-Tactin®. Strep-tag®II binds to the biotin binding pocket enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the Strep-Tactin® affinity resins. Fast binding and dissociation kinetics allow comparatively high flow rates throughout column chromatography including elution. Fast dissociation kinetics may be, however, limiting for using Strep-tag®II for direct purification of target proteins from large volumes of diluted extracts like mammalian cell culture supernatants or in assay formats requiring extended washing like ELISA. For this reason, binding characteristics were improved by development of the Twin-Strep-tag® consisting of two Strep-tag®II moieties connected by a short linker. The resulting avidity effect, i.e., the combined synergistic binding of two Strep-tag®II moieties to tetrameric Strep-Tactin®, reduces the off-rate for more steady binding under non-competitive conditions. The addition of a competitor, however, reverses the synergistic avidity effect and, hence, efficient elution capability is preserved. In fact, the Twin-Strep-tag® features all beneficial properties of Strep-tag®II, including efficient elution under gentle competitive conditions, but, due to its higher affinity, additionally enables a more universal use in applications requiring stable binding.

Keywords: Affinity purification; Avidity; Cell culture; Recombinant protein expression; Twin-Strep-tag®.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biotin / metabolism
  • Cell Line
  • Chromatography, Affinity / methods*
  • Humans
  • Molecular Sequence Data
  • Oligopeptides / chemistry
  • Oligopeptides / isolation & purification
  • Oligopeptides / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification*
  • Recombinant Fusion Proteins / metabolism*
  • Streptavidin / metabolism


  • Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly
  • Oligopeptides
  • Recombinant Fusion Proteins
  • Biotin
  • Streptavidin