Strep-tag® - Leading affinity chromatography system
From purification to analytical applications
Our proprietary Strep-tag® technology exploits one of the strongest non-covalent interactions in nature: the interaction of biotin and streptavidin. The system is based on the highly selective and easily controllable interaction between the synthetic Strep-tag®II peptide and the specially engineered streptavidin, called Strep-Tactin®, which is one of the most stable proteins known. The Strep-tag®II binds specifically to the engineered streptavidins, Strep-Tactin® and Strep-Tactin®XT, by occupying the binding pocket of the natural ligand biotin. Hence, the interaction is easily reversible by excessive addition of the competitor.
Two affinity tags – two streptavidin derivatives: The Strep-tag®II consists of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), whereas the Twin-Strep-tag® includes this motif two times in series connected by a linker and is accordingly composed of 28 amino acids. Both exhibit intrinsic, although unequal, affinity towards the streptavidin derivative Strep-Tactin® and its successor Strep-Tactin®XT: The binding affinity of Strep-tag®II to Strep-Tactin® (1µM) is nearly 100 times higher than to streptavidin. A further improvement was achieved by the development of Strep-Tactin®XT, which shares a nM affinity with the Strep-tag®II and a pM affinity with the Twin-Strep-tag®.
The Strep-tag® protein purification system comprises two affinity tags, the 8 aa Strep-tag®II and its tandem version Twin-Strep-tag®. Both versions can bind to Strep-Tactin® and its high affinity variant Strep-Tactin®XT. Thereby the two tags differ in the affinities with which they bind to Strep-Tactin® and Strep-Tactin®XT. Depending on the application and properties of the protein of interest one can combine the different tags and Strep-Tactin® variants according to the required affinity.
As a result of the differences in binding strength among the possible tag-ligand combinations, the Strep-tag® system has become established as a universal tool for isolation of proteins and cells.
- Fused to recombinant proteins, the Strep-tag® enables efficient one-step purification on immobilized Strep-Tactin®.
- When fused to antibody-derived Fab fragments or nanobodies, Twin-Strep-tag® binds to multimerized Strep-Tactin® allowing capturing and releasing of target cells based on their surface-marker or antigen-specificity.
- Strep-Tactin® (or Strep-Tactin®XT), on its turn, can be conjugated to microplates, fluorophores or chips, allowing a wide range of analytical applications after isolation of target material, such as detection, immobilization and interaction studies.
Key features of the Strep-tag® system are:
- Reversibility
- Full functionality of target material after isolation
- High purity
- Universal applicability for various analytic applications
- Simple purification procedures
Which resin to choose when purifying strep-tagged proteins?
Strep-Tactin® and its high-affinity counterpart, Strep-Tactin®XT, are both streptavidin-derived mutants. Each of these proteins weighs 52 kDa, comprising four subunits, each housing a biotin binding pocket. While streptavidin binds biotin with an almost irreversible affinity in its pocket, Strep-Tactin® and Strep-Tactin®XT exhibit reduced affinity for biotin. However, they still maintain the capability to bind biotin. The biotin binding pockets on both Strep-Tactin® and Strep-Tactin®XT have been optimized for binding with Strep-tag® and its tandem version, Twin-Strep-tag®. In contrast to the near irreversibility of biotin binding with streptavidin, the interaction with either tag on Strep-Tactin® or Strep-Tactin®XT is reversible. Compared to streptavidin, a crucial alteration in Strep-Tactin® involves mutating the lid-like loop, resulting in a consistently open conformation of the loop. This initial mutation enhances the affinity for strep-tagged proteins by a factor of ten. Upon peptide binding, the lid remains open, allowing for easy removal of the peptide by competitive biotin. In Strep-Tactin®XT, a second mutation was introduced on the opposite side of the chain. This alteration enhances the interaction between the peptide and the biotin binding pocket, significantly boosting the binding affinity. More detail about the improvements of the sequence can be found in Schmidt et al. 2021.
