Strep-tag II

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The Strep -tag II is a protein tag that enables the purification and detection of recombinant proteins by means of affinity chromatography . It is a synthetic peptide , which consists of 8 amino acids and can be expressed N- or C-terminally with a fusion protein. The Strep -tag II has a strong affinity for Strep -actin, which is used for the purification of the fusion proteins on affinity chromatography columns. In this method, the Strep -tag II fusion proteins are eluted by adding a biotin derivative that competes with the Strep -tag II. One advantage of Strep -tag is the mild physiological conditions under which protein purification takes place. Because of this, the Strep -tag system is particularly suitable for the expression of bioactive, functional proteins.

Origin of the Strep tag

The long-known bond between streptavidin and biotin ( vitamin H ) served as the basis for the development of the Strep -tag . Streptavidin is a protein from the bacterium Streptomyces avidinii , which consists of four identical subunits . Each of these subunits can bind a molecule of biotin. This bond has a high affinity and represents one of the strongest known, non-covalent bonds. Because of this property, streptavidin is often used in molecular biology , biotechnology and biochemistry .

The Strep -tag is a peptide that was developed for binding in the biotin binding pocket of streptavidin in order to be able to serve as a tool for recombinant protein purification. The later further development is the Strep -tag II (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), which in turn is characterized by an improved binding to Strep -Tactin - a modified streptavidin variant. The affinity of Strep -tag II for Strep -Tactin is about 10 times higher than that for streptavidin. The Strep -tag system optimized in this way , consisting of Strep -tag II and Strep -Tactin, has proven to be extremely useful for the isolation of functional proteins and protein complexes, as well as their detection in the context of proteome studies.

The principle of the Strep -tag system

The Strep -tag is introduced into a vector by cloning in front of or behind the gene sequence of the desired protein , whereby a fusion protein with the N- or C-terminal Strep -tag II is produced during the expression . Vectors for various host organisms are available for expression. In addition to vectors for expression in E. coli, there are also vectors available for yeasts and for insect and mammalian cells. Since the Strep -tag II is only small in size and almost biochemically inert , it does not affect protein folding or secretion and does not affect the function of the protein. Another advantage is that the purification steps can be carried out under physiological conditions, which ensures the production of biologically active proteins and allows the purification of functional protein complexes. A comparison with other protein tags showed a very high purity (> 95%) and a high yield of purified protein for Strep -tag II.

The purification of Strep -tag II fusion proteins begins with the cell lysate with the fusion protein being applied to a column with immobilized Strep -tactin. The fusion protein then binds to Strep -Tactin. This is followed by a washing step in which all unbound proteins are washed from the column with a physiological buffer (e.g. PBS ) . The fusion protein is then eluted with a low concentration of desthiobiotin, an analogue of biotin , the natural ligand of streptavidin . Desthiobiotin competes specifically for the biotin binding site with high affinity. It has the advantage over biotin that it binds to Strep- Tactin with less affinity and therefore the matrix can be regenerated.

In order to regenerate the Strep -Tactin matrix, desthiobiotin is displaced from the column by adding a solution that contains an excess of the yellow azo dye HABA , which binds to Strep -Tactin . The binding of HABA to Strep- Tactin leads to a color change in the matrix from yellow to orange-red and thus makes the degree of regeneration visible. Finally, the bound HABA is also removed from the column by adding washing buffer. Complete regeneration takes place when the column has returned to its original white color. Then it can be used for the next cleaning.

Applications

The Strep -tag system enables protein purification by means of affinity chromatography under physiological conditions with high specificity. The purified proteins retain their biological activity and are then in a very pure form (over 95% purity). The purification of protein complexes is also possible with this method, since the complexes are retained in the physiological buffer.

In addition, the Strep -tag II can be used for the detection of proteins in different assays. For example, antibodies against Strep -tag II can be used for detection by means of immunofluorescence , FACS , ELISA and Western blots . There are also strep- tactin conjugates with enzymatic (e.g. horseradish peroxidase HRP; alkaline phosphatase AP) or fluorescent markers that can be used for the direct detection of fusion proteins with Strep -tag II. In this case, detection can be carried out via immunofluorescence , Western blots or dot blots .

In addition, the can Strep -tag system for identifying protein-protein interactions (eg. B. pull-down assays ), as well as for immobilization of Strep -tag fusion protein to microtiter plates or biochips are used for further interaction analyzes.

Overview of the application possibilities of the Strep -tag system:

Individual evidence

  1. ↑ a b A Skerra: The Strep -tag as a molecular tool for high-throughput protein purification in proteome research . In: BIOspectrum . 2, 2003, pp. 189-192.
  2. ↑ a b A Skerra, T Schmidt: Use of the Strep Tag and streptavidin for detection and purification of recombinant proteins . In: Methods in Enzymology . 326, 2000, pp. 271-304.
  3. ↑ WA Hendrickson: Crystal Structure of Core Streptavidin Determined from Multiwavelength Anomalous Diffraction of Synchrotron Radiation . In: Proceedings of the National Academy of Sciences . 86 (7), 1989, pp. 2190-4.
  4. ^ A b c T Schmidt, A Skerra: The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins . In: Nature Protocols . 2 (6), 2007, pp. 1528-35.
  5. ↑ M Junttila, S Saarinen, T Schmidt, J Kast, J Westermarck: Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells . In: Proteomics . 5, 2005, pp. 1199-1203.
  6. Jump up ↑ JJ Lichty, J Malecki, HD Agnew, HJ Michelson-Horowitz, S Tan: Comparison of affinity tags for protein purification . In: Protein Expr Purif . 41, 2005, pp. 98-105.
  7. ↑ a b J D Hirsch, L Eslamizar, B Filanoski, N Malekzadeh, RP Haugland, JM Beechem, RP Haugland: Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding: uses for protein labeling, detection, and isolation proteins . In: Anal Biochem . 308 (2), 2002, pp. 343-57.
  8. ↑ SN Thulasi Raman, Y Zhou: Networks of Host Factors that Interact with NS1 Protein of Influenza A Virus . In: Front Microbiol . 7: 654, 2016.
  9. ↑ NP Hoe, E Lukomska, JM Musser, S Lukomsky: Characterization of the immune response to collagen-like proteins SCL1 and ACL2 of serotype M1 and M28 group A Streptococcus . In: FEMS Microbiol Lett . 277, 2007, pp. 142-149.
  10. ↑ C Lorenz, D Büttner: Functional Characterization of the Type III Secretion ATPase HrcN from the Plant Pathogen Xanthomonas campestris pv. Vesicatoria . In: J Bacteriol . 151 (5), 2008, pp. 1414-1428.
  11. ↑ D Sermwittayawong, S Tan: SAGA binds TBP via its Spt8 subunit in competition with DNA: implications for TBP recruitment . In: EMBO J . 25, 2006, pp. 3791-3800.