Strep-tag
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The Strep-tag system is a method which allows the purification and detection of
Development and biochemistry of the Strep-tag
Streptavidin is a tetrameric protein expressed in Streptomyces avidinii. Because of Streptavidin's high affinity for vitamin H (biotin), Streptavidin is commonly used in the fields of molecular biology and biotechnology. The Strep-tag was originally selected from a genetic library to specifically bind to a proteolytically truncated "core" version of streptavidin. Over the years, the Strep-tag was systemically optimized, to permit a greater flexibility in the choice of attachment site. Further, its interaction partner, Streptavidin, was also optimized to increase peptide-binding capacity, which resulted in the development of Strep-Tactin. The binding affinity of Strep-tag to Strep-Tactin is nearly 100 times higher than from Strep-tag to Streptavidin. The so-called Strep-tag system, consisting of Strep-tag and Strep-Tactin, has proven particularly useful for the functional isolation and analysis of protein complexes in proteome research.[3]
The Strep-tag principle
Just like other short-affinity tags (
In the first step of the Strep-tag purification cycle, the
Strep-tag applications
The Strep-tag system offers a selective tool to purify proteins under physiological conditions. The proteins obtained are bioactive and display a very high purity (above 95%). Also, the Strep-tag system can be used for protein detection in various assays. Depending on the experimental circumstances, Strep-tag antibodies or Strep-Tactin, with an enzymatic (e.g.horseradish peroxidase (HRP), alkaline phosphatase (AP)) or fluorescence (e.g. green fluorescent protein (GFP)) marker. If high purity is required, the lysate can be purified by first using Strep-Tactin and then perform a second run using antibodies against Strep-tag. This reduces the contamination with unspecific bound proteins, which might occur in some rare scenarios.
Following assays can be conducted using the Strep-tag detection system:
- one-step affinity purification
- Protein:protein interaction studies
- Colony blot, dot blot, Western blot and ELISA
- Screening for positive expression clones
- Immunocytochemistry and Immunohistochemistry
- Protein localization and targeting studies
Because the Strep-tag is capable of isolating protein complexes, strategies for the study of protein-protein interactions can also be conducted. Another option is the immobilization of Strep-tag proteins with a specific high affinity antibody on microplates or biochips.
Strep-Tag/StrepTactin system is also used in
See also
References
- S2CID 25209313.
- PMID 11036648.
- PMID 9380672.
- ^ https://www.qiagen.com/resources/download.aspx?id=0fadc040-86ad-4e31-bb58-7e83ceb77b72&lang=en [bare URL PDF]
- S2CID 24773674.
- ^ Moayed F, Mashaghi A, Tans SJ (2013) A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers. PLoS ONE 8(1): e54440. doi:10.1371/journal.pone.0054440 [1]