Strep-tag®: The leading affinity tag in recombinant protein technology

Strep-tag^® principle

The basis for the development of the Strep-tag^® system is the well known binding of biotin to streptavidin. To take advantage of this very strong interaction in protein purification applications it was necessary to investigate 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. The found peptid was a short sequence consisting of only 8 amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) and was named Strep-tag^®II.

In order to optimize the binding properties, also streptavidin has 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.

Strep-tag^®II

The short peptide tag (8 amino acids) has only negligible effects 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.

Benefits of Strep-tag^®II:

• it does not interfere with folding or bioactivity
• does not react with heavy metal ion buffer impurities
• has no ion exchange properties
• does not induce protein aggregation
• no need for removing the tag

Strep-Tactin^®

Strep-Tactin^® is a derivative of streptavidin, which is one of the most stable proteins known.

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: 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^®II 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