SBP-tag
Lua error in package.lua at line 80: module 'strict' not found. The Streptavidin-Binding Peptide (SBP)-Tag is a 38-amino acid sequence that may be engineered into recombinant proteins. Recombinant proteins containing the SBP-Tag bind to streptavidin and this property may be utilized in specific purification, detection or immobilization strategies.[citation needed]
The sequence of the SBP tag is MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP.[1]
Contents
Discovery
The Streptavidin-Binding Peptide was discovered within a library of seven trillion stochastically-generated peptides using the in vitro selection technique of mRNA Display. Selection was performed by incubating with streptavidin-agarose followed by elution with biotin.[2] The SBP-Tag has been shown to bind streptavidin with an equilibrium dissociation constant of 2.5nM[1][2] and is readily eluted with biotin under native conditions.[1][2]
Applications
Protein purification
Because of the mild elution conditions (biotin plus wash buffer) SBP-Tagged proteins can be generated in a relatively pure state with a single purification step.[1][3][4] There are several relatively abundant mammalian proteins that inherently associate with the IMAC matrices that bind to the more commonly used Polyhistidine-tag (His-tag). For this reason non-IMAC purification protocols, including with the SBP-Tag, are often preferred for proteins that are expressed in mammalian cells.[citation needed]
Protein complex purification
Complexes of interacting proteins may also be purified using the SBP-Tag because elution with biotin permits recovery under conditions in which desired complexes remain associated. For example, the Condensin Complex was purified by Kim et al. [2010] and complexes with the TAZ transcriptional co-activator were purified by Zhang et al. [2009]. The SBP-Tag has also been incorporated into several Tandem Affinity Purification (TAP) systems in which successive purification steps are utilized with multiple tags, for example GFP fusion proteins and BTK-protein complexes were purified using a TAP protocol with the SBP-Tag and the His-Tag,[5][6] HDGF-protein complexes were purified using a TAP protocol with the SBP-Tag and with the FLAG-tag[7] and Wnt complexes were purified using a TAP protocol with the SBP-Tag and with the [Calmodulin-Tag].[8] TAP is generally used with protein complexes and several studies report significant improvements in purity and yield when the SBP-Tag TAP systems are compared to non-SBP-Tag systems.[9][10][11] Commercial TAP systems that use the SBP-Tag include the Interplay® Adenoviral and Mammalian TAP Systems sold by Agilent Technologies, similar products are sold by Sigma-Aldrich.[12]
Proteomics
Screens for biologically relevant protein-protein interactions have been performed using Tandem Affinity Purification (TAP) with the SBP-Tag and Protein A,[10] for interaction proteomics and transcription factor complexes with the SBP-Tag and Protein G,[10][13] for proteins that interact with the Dengue Virus protein DENV-2 NS4A with the SBP-Tag and the Calmodulin Tag.[14] and for proteins that interact with protein phosphatase 2A (PP2A) with the SBP-Tag and the hemagglutinin (HA)-tag.[11]
Imaging
The SBP-Tag will also bind to streptavidin or streptavidin reagents in solution. Applications of these engineered associations include the visualization of specific proteins within living cells,[15] monitoring of the kinetics of the translation of individual proteins in an in vitro translation system,[16] control of the integration of a multi-spanning membrane protein into the endoplasmic reticulum by fusing the SBP-Tag to the N-terminal translocation sequence and then halting integration with streptavidin and restarting integration with biotin.[17][18] Fluorescent streptavidin reagents (e.g. streptavidin-HRP) can be used to visualize the SBP-tag by immunoblotting of SDS-PAGE.[1][19][20] Additionally, antibodies to the SBP-tag are available commercially.[citation needed]
Surface plasmon resonance
The SBP-Tag has been used to reversibly immobilize recombinant proteins onto streptavidin-functionalized surfaces thereby permitting interaction assessment such as by surface plasmon resonance (SPR) techniques with re-use of the functionalized surface.[21] SPR has also been used to compare the SBP-Tag with other streptavidin-binding peptides such as Strep-tag.[22]
See also
References
- ↑ 1.0 1.1 1.2 1.3 1.4 Lua error in package.lua at line 80: module 'strict' not found.
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Further reading
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