Strep-tag® - The leading affinity tag in recombinant protein technology
The Strep-tag® - Strep-Tactin® system developed by IBA, is one of the most widely used affinity chromatography systems in protein purification, detection and immobilization, and offers many advantages:
- Strep-tag® II undisturbing 8 amino acids short tag
- Rapid one-step purification under physiological conditions
- Unsurpassed purity and bioactivity
- Balanced amino acid composition - generally no effect on protein structure or activity
- Highly selective and easily controllable binding properties
- C- or N-terminal fusion
- Removal not required
Strep-tag® is the method of choice for:
- membrane proteins
- sensitive protein complexes with multiple subunits
- and any other protein (see references)!
For the use of this system we offer a large range of high quality products, such as
- Cloning/Expression Vectors for different hosts
- Various Starter Kits for newcomers
- Different Strep-Tactin® purification resins and pre-packed columns
- Detection Systems with Strep-Tactin® conjugates or a monoclonal antibody
- His-STREPPER conversion tool - to convert a His-tag fusion protein into a Strep-tag fusion protein without cloning
IBA has developed a universal platform called Strep-tag®. This platform was developed in close cooperation with Prof. Dr. Arne Skerra, TU Munich, for the rapid and cost effective as well as versatile production and use of recombinant proteins.
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.
To optimize binding properties, streptavidin has also been engineered to obtain Strep-Tactin®. Thus, the optimal binding partners have been found: The Strep-tag® / Strep-Tactin® system is now one of the most widely used affinity chromatography systems.
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® 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.
- 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: Bound Strep-tag®II fusion proteins are then gently eluted by adding wash buffer supplemented with 2.5 mM desthiobiotin (Buffer E), which specifically competes for the biotin binding pocket.
Since the buffer conditions during elution essentially remain unchanged, potentially unspecifically bound 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 color has disappeared the column can be re-used. Strep-Tactin® resin can be regenerated and re-used 3 to 5 times without loss of performance.
Purification of a GFP-Strep-tag® II fusion protein, which has been overexpressed in E. coli .
Pictures left to right:1, New or regenerated column; 2, specific binding of GFP-Strep-tag® II fusion protein to Strep-Tactin® Sepharose® column while unspecific proteins are rapidly washed away with small amounts of physiological buffer; 3, Strep-tag® protein is eluted due to addition of the specific competitor "desthiobiotin"; 4 to 6, column regeneration: desthiobiotin is displaced by the yellow solution HABA, which turns red once complexed with Strep-Tactin®. HABA is then removed by washing buffer and the column can be re-used.