SBP-tag

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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 ]

Contents

The sequence of the SBP tag is MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP. [1]

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. 1 2 3 4 5 Keefe, Anthony D.; Wilson, David S.; Seelig, Burckhard; Szostak, Jack W. (2001). "One-Step Purification of Recombinant Proteins Using a Nanomolar-Affinity Streptavidin-Binding Peptide, the SBP-Tag". Protein Expression and Purification. 23 (3): 440–6. doi:10.1006/prep.2001.1515. PMID   11722181.
  2. 1 2 3 Wilson, David S.; Keefe, Anthony D.; Szostak, Jack W. (2001). "The use of mRNA display to select high-affinity protein-binding peptides". Proceedings of the National Academy of Sciences. 98 (7): 3750–5. Bibcode:2001PNAS...98.3750W. doi: 10.1073/pnas.061028198 . PMC   31124 . PMID   11274392.
  3. ↑ Ichikawa, Muneyoshi; Watanabe, Yuta; Murayama, Takashi; Toyoshima, Yoko Yano (2011). "Recombinant human cytoplasmic dynein heavy chain 1 and 2: Observation of dynein-2 motor activity in vitro". FEBS Letters. 585 (15): 2419–23. doi:10.1016/j.febslet.2011.06.026. PMID   21723285. S2CID   27909093.
  4. ↑ Li, Feng; Herrera, Jeremy; Zhou, Sharleen; Maslov, Dmitri A.; Simpson, Larry (2011). "Trypanosome REH1 is an RNA helicase involved with the 3'-5' polarity of multiple gRNA-guided uridine insertion/deletion RNA editing". Proceedings of the National Academy of Sciences. 108 (9): 3542–7. Bibcode:2011PNAS..108.3542L. doi: 10.1073/pnas.1014152108 . PMC   3048136 . PMID   21321231.
  5. ↑ Li, Yifeng; Franklin, Sarah; Zhang, Michael J.; Vondriska, Thomas M. (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. 20 (1): 140–9. doi:10.1002/pro.546. PMC   3047070 . PMID   21080425.
  6. ↑ Kobayashi, Takuya; Morone, Nobuhiro; Kashiyama, Taku; Oyamada, Hideto; Kurebayashi, Nagomi; Murayama, Takashi (2008). Imhof, Axel (ed.). "Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research". PLOS ONE. 3 (12) e3822. Bibcode:2008PLoSO...3.3822K. doi: 10.1371/journal.pone.0003822 . PMC   2585475 . PMID   19048102.
  7. ↑ Zhao, Jian; Yu, Hongxiu; Lin, Ling; Tu, Jun; Cai, Lili; Chen, Yanmei; Zhong, Fan; Lin, Chengzhao; et al. (2011). "Interactome study suggests multiple cellular functions of hepatoma-derived growth factor (HDGF)". Journal of Proteomics. 75 (2): 588–602. doi:10.1016/j.jprot.2011.08.021. PMID   21907836.
  8. ↑ Ahlstrom, Robert; Yu, Alan S. L. (2009). "Characterization of the kinase activity of a WNK4 protein complex". AJP: Renal Physiology. 297 (3): F685–92. doi:10.1152/ajprenal.00358.2009. PMC   2739714 . PMID   19587141.
  9. ↑ Kyriakakis, Phillip P.; Tipping, Marla; Abed, Louka; Veraksa, Alexey (2008). "Tandem affinity purification in Drosophila: The advantages of the GS-TAP system". Fly. 2 (4): 229–35. doi: 10.4161/fly.6669 . PMID   18719405.
  10. 1 2 3 Bürckstümmer, Tilmann; Bennett, Keiryn L; Preradovic, Adrijana; Schütze, Gregor; Hantschel, Oliver; Superti-Furga, Giulio; Bauch, Angela (2006). "An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells". Nature Methods. 3 (12): 1013–9. doi:10.1038/nmeth968. PMID   17060908. S2CID   7069058.
  11. 1 2 Glatter, Timo; Wepf, Alexander; Aebersold, Ruedi; Gstaiger, Matthias (2009). "An integrated workflow for charting the human interaction proteome: Insights into the PP2A system". Molecular Systems Biology. 5 (1): 237. doi:10.1038/msb.2008.75. PMC   2644174 . PMID   19156129.
  12. ↑ Li, Yifeng (2011). "The tandem affinity purification technology: An overview". Biotechnology Letters. 33 (8): 1487–99. doi:10.1007/s10529-011-0592-x. PMID   21424840. S2CID   157683.
  13. ↑ Van Leene, Jelle; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Geerinck, Jan; Van Isterdael, Gert; Witters, Erwin; De Jaeger, Geert (2011). "Isolation of Transcription Factor Complexes from Arabidopsis Cell Suspension Cultures by Tandem Affinity Purification". In Yuan, Ling; Perry, Sharyn E (eds.). Plant Transcription Factors. Methods in Molecular Biology. Vol. 754. pp. 195–218. doi:10.1007/978-1-61779-154-3_11. ISBN   978-1-61779-153-6. PMID   21720954.
  14. ↑ Anwar, Azlinda; Leong, K. M.; Ng, Mary L.; Chu, Justin J. H.; Garcia-Blanco, Mariano A. (2009). "The Polypyrimidine Tract-binding Protein Is Required for Efficient Dengue Virus Propagation and Associates with the Viral Replication Machinery". Journal of Biological Chemistry. 284 (25): 17021–9. doi: 10.1074/jbc.M109.006239 . PMC   2719340 . PMID   19380576.
  15. ↑ McCann, Corey M.; Bareyre, Florence M.; Lichtman, Jeff W.; Sanes, Joshua R. (2005). "Peptide tags for labeling membrane proteins in live cells with multiple fluorophores". BioTechniques. 38 (6): 945–52. doi: 10.2144/05386IT02 . PMID   16018556.
  16. ↑ Takahashi, Shuntaro; Iida, Masaaki; Furusawa, Hiroyuki; Shimizu, Yoshihiro; Ueda, Takuya; Okahata, Yoshio (2009). "Real-Time Monitoring of Cell-Free Translation on a Quartz-Crystal Microbalance". Journal of the American Chemical Society. 131 (26): 9326–32. doi:10.1021/ja9019947. PMID   19518055.
  17. ↑ Kida, Yuichiro; Morimoto, Fumiko; Sakaguchi, Masao (2007). "Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis". The Journal of Cell Biology. 179 (7): 1441–52. doi:10.1083/jcb.200707050. PMC   2373506 . PMID   18166653.
  18. ↑ Kida, Y.; Morimoto, F.; Sakaguchi, M. (2008). "Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane". Journal of Biological Chemistry. 284 (5): 2861–6. doi: 10.1074/jbc.M808020200 . PMID   19010775.
  19. ↑ Edelmann, Mariola J.; Iphöfer, Alexander; Akutsu, Masato; Altun, Mikael; Di Gleria, Katalin; Kramer, Holger B.; Fiebiger, Edda; Dhe-Paganon, Sirano; Kessler, Benedikt M. (2009). "Structural basis and specificity of human otubain 1-mediated deubiquitination" (PDF). Biochemical Journal. 418 (2): 379–90. doi:10.1042/BJ20081318. PMID   18954305.
  20. ↑ Hoer, Simon; Smith, Lorraine; Lehner, Paul J. (2007). "MARCH-IX mediates ubiquitination and downregulation of ICAM-1". FEBS Letters. 581 (1): 45–51. doi:10.1016/j.febslet.2006.11.075. PMID   17174307. S2CID   22461058.
  21. ↑ Li, Yong-Jin; Bi, Li-Jun; Zhang, Xian-En; Zhou, Ya-Feng; Zhang, Ji-Bin; Chen, Yuan-Yuan; Li, Wei; Zhang, Zhi-Ping (2006). "Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip". Analytical and Bioanalytical Chemistry. 386 (5): 1321–6. doi:10.1007/s00216-006-0794-6. PMID   17006676. S2CID   6074268.
  22. ↑ Huang, Xu; Zhang, Xian-En; Zhou, Ya-Feng; Zhang, Zhi-Ping; Cass, Anthony E. G. (2007). "Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides". Journal of Biochemical and Biophysical Methods. 70 (3): 435–9. doi:10.1016/j.jbbm.2006.10.006. PMID   17156847.

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