SBP-tag

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. The SBP-Tag has been shown to bind streptavidin with an equilibrium dissociation constant of 2.5nM and is readily eluted with biotin under native conditions.

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. 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.

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, HDGF-protein complexes were purified using a TAP protocol with the SBP-Tag and with the FLAG-tag and Wnt complexes were purified using a TAP protocol with the SBP-Tag and with the [Calmodulin-Tag]. 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. 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.

Proteomics

Screens for biologically relevant protein-protein interactions have been performed using Tandem Affinity Purification (TAP) with the SBP-Tag and Protein A, for interaction proteomics and transcription factor complexes with the SBP-Tag and Protein G, for proteins that interact with the Dengue Virus protein DENV-2 NS4A with the SBP-Tag and the Calmodulin Tag. and for proteins that interact with protein phosphatase 2A (PP2A) with the SBP-Tag and the hemagglutinin (HA)-tag.

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, monitoring of the kinetics of the translation of individual proteins in an in vitro translation system, 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. Fluorescent streptavidin reagents (e.g. streptavidin-HRP) can be used to visualize the SBP-tag by immunoblotting of SDS-PAGE. Additionally, antibodies to the SBP-tag are available commercially.

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. SPR has also been used to compare the SBP-Tag with other streptavidin-binding peptides such as Strep-tag.

References

References

1. (2001). "The use of mRNA display to select high-affinity protein-binding peptides". Proceedings of the National Academy of Sciences.
2. (2001). "One-Step Purification of Recombinant Proteins Using a Nanomolar-Affinity Streptavidin-Binding Peptide, the SBP-Tag". Protein Expression and Purification.
3. (2011). "Recombinant human cytoplasmic dynein heavy chain 1 and 2: Observation of dynein-2 motor activity in vitro". FEBS Letters.
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5. (2011). "Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase". Protein Science.
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8. (2009). "Characterization of the kinase activity of a WNK4 protein complex". AJP: Renal Physiology.
9. (2008). "Tandem affinity purification in Drosophila: The advantages of the GS-TAP system". Fly.
10. (2006). "An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells". Nature Methods.
11. (2009). "An integrated workflow for charting the human interaction proteome: Insights into the PP2A system". Molecular Systems Biology.
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14. (2009). "The Polypyrimidine Tract-binding Protein Is Required for Efficient Dengue Virus Propagation and Associates with the Viral Replication Machinery". Journal of Biological Chemistry.
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16. (2009). "Real-Time Monitoring of Cell-Free Translation on a Quartz-Crystal Microbalance". Journal of the American Chemical Society.
17. (2007). "Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis". The Journal of Cell Biology.
18. (2008). "Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane". Journal of Biological Chemistry.
19. (2009). "Structural basis and specificity of human otubain 1-mediated deubiquitination". Biochemical Journal.
20. (2007). "MARCH-IX mediates ubiquitination and downregulation of ICAM-1". FEBS Letters.
21. (2006). "Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip". Analytical and Bioanalytical Chemistry.
22. (2007). "Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides". Journal of Biochemical and Biophysical Methods.