Strep-tag® principle

IBA has developed a universal platform called Strep-tag®. This platform for the rapid and cost effective as well as versatile production and use of recombinant proteins was developed in close cooperation with Prof. Dr. Arne Skerra, TU Munich. 

The basis for the development of the Strep-tag® principle was the well known binding of biotin to streptavidin. To take advantage of this strong interaction in protein purification applications we found it desirable to have a peptide that is capable of binding to the biotin binding pocket of streptavidin when fused to recombinant proteins. This peptide was supposed to serve as purification tag. Finally, we succeeded in engineering a short sequence consisting of only 8 amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) and named it Strep-tag® II.

The Strep-tag® purification system is based on the highly selective and easily controllable interaction between the Strep-tag® II peptide and specially engineered streptavidin called Strep-Tactin®. The binding affinity of Strep-tag® II to Strep-Tactin® is nearly 100 times higher than to streptavidin. The tagged protein binds to immobilized Strep-Tactin® during affinity purification. Physiological buffers like PBS in combination with a wide range of additives can be used (see Table 1, Strep-Tactin® resin specifications). After a short washing step, gentle elution of purified recombinant protein is performed by addition of desthiobiotin (2.5 mM) in the same buffer. Desthiobiotin is an inexpensive, reversibly binding and stable analog of biotin – the natural ligand of streptavidin. This competitive elution is the second step conferring specificity thus enabling unparalleled purification factors. The system is safe and easy to use; column regeneration and activity status are visualized by a color change on the purification column.

Strep-tag®II

The short peptide tag (8 amino acids) has negligible effect on the recombinant protein due to its chemically balanced amino acid composition (WSHPQFEK). The tag can be placed at the C- or N-terminus. A two amino acid spacer between the protein and the tag is recommended to ensure accessibility of the tag. Generally, it does not interfere with folding or bioactivity, does not react with heavy metal ion buffer impurities, has no ion exchange properties and does not induce protein aggregation. Thus, there is no need for removing the tag.

Strep-Tactin®

Strep-Tactin® is a streptavidin derivative which is one of the most stable proteins known. Streptavidin is stable to treatment with 8 M urea or guanidine, 0.5 M NaOH as well as 50 % formamide (t = 1 h; T = 37 °C). Proteases (proteinase pepsin, papain, subtilisin, thermolysin, elastase) do not cleave streptavidin during a 2 h incubation at a 1:50 w/w ratio and 37 °C. In the presence of SDS streptavidin begins to break up into monomers only at temperatures above 60 °C. As far as tested, we have been able to confirm these extraordinary properties for Strep-Tactin®, thus enabling long-lasting affinity columns which can be re-used 3-5 times. Furthermore, the neutral pI of Strep-Tactin® minimizes non-specific protein or nucleic acid binding.

Benefits

• Purification of bioactive recombinant proteins
• Physiological purification using desthiobiotin elution
• Protein aggregation is avoided
• Broad range of detergents, chelators, salt or redox conditions allowed
• Avoids interaction with heavy metal ions which are toxic and may catalyze protein oxidation

Purification procedure under physiological conditions

The purification of Strep-tag® II fusion proteins is easy, straightforward and user-friendly. The complete procedure can be performed under nearly physiological conditions, e.g. in PBS buffer and for elution in PBS/2.5 mM desthiobiotin buffer:

Step 3: Then, bound Strep-tag® II protein is gently eluted by adding wash buffer containing additionally 2.5 mM desthiobiotin (Buffer E) which specifically competes for the biotin binding pocket.

Since the buffer conditions during elution essentially remain unchanged, potentially unspecific binding proteins (without Strep-tag®) will not be eluted and, thus, will not contaminate the protein of interest. Next to the specific binding of Strep-tag® to Strep-Tactin®, this is the second specificity conferring step of this purification procedure, yielding extremely high protein purity.

Steps 4: To regenerate the column the yellow azo dye HABA (2- [4’-hydroxy-benzeneazo] benzoic acid) is added (buffer R) in excess to displace desthiobiotin from the binding pocket. Once HABA binds to the binding site, the color turns to red conveniently indicating the regeneration and activity status of the column.

Step 5: HABA can be removed simply by adding wash buffer. Once the red colour has disappeared the column can be re-used. Strep-Tactin® resin can be regenerated and re-used 3 to 5 times without loss in performance.