SBP-tag

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|date=November 2011}}

The sequence of the SBP tag is MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP.

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.{{cite journal |pages=3750–5 |doi=10.1073/pnas.061028198 |pmc=31124 |title=The use of mRNA display to select high-affinity protein-binding peptides |year=2001 |last1=Wilson |first1=David S. |first2=Anthony D. |last2=Keefe |first3=Jack W. |last3=Szostak |journal=Proceedings of the National Academy of Sciences |volume=98 |issue=7 |pmid=11274392|bibcode=2001PNAS...98.3750W |doi-access=free }} The SBP-Tag has been shown to bind streptavidin with an equilibrium dissociation constant of 2.5nM{{cite journal |pages=440–6 |doi=10.1006/prep.2001.1515 |title=One-Step Purification of Recombinant Proteins Using a Nanomolar-Affinity Streptavidin-Binding Peptide, the SBP-Tag |year=2001 |last1=Keefe |first1=Anthony D. |journal=Protein Expression and Purification |volume=23 |issue=3 |pmid=11722181 |last2=Wilson |first2=David S. |last3=Seelig |first3=Burckhard |last4=Szostak |first4=Jack W.}} 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.{{cite journal |pages=2419–23 |doi=10.1016/j.febslet.2011.06.026 |title=Recombinant human cytoplasmic dynein heavy chain 1 and 2: Observation of dynein-2 motor activity in vitro |year=2011 |last1=Ichikawa |first1=Muneyoshi |last2=Watanabe |first2=Yuta |last3=Murayama |first3=Takashi |last4=Toyoshima |first4=Yoko Yano |journal=FEBS Letters |volume=585 |issue=15 |pmid=21723285|s2cid=27909093 }}{{cite journal |pages=3542–7 |doi=10.1073/pnas.1014152108 |pmc=3048136 |title=Trypanosome REH1 is an RNA helicase involved with the 3'-5' polarity of multiple gRNA-guided uridine insertion/deletion RNA editing |year=2011 |last1=Li |first1=Feng |last2=Herrera |first2=Jeremy |last3=Zhou |first3=Sharleen |last4=Maslov |first4=Dmitri A. |last5=Simpson |first5=Larry |journal=Proceedings of the National Academy of Sciences |volume=108 |issue=9 |pmid=21321231|bibcode=2011PNAS..108.3542L |doi-access=free }} 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|date=November 2011}}

=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,{{cite journal |pages=140–9 |doi=10.1002/pro.546 |pmc=3047070 |title=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 |year=2011 |last1=Li |first1=Yifeng |last2=Franklin |first2=Sarah |last3=Zhang |first3=Michael J. |last4=Vondriska |first4=Thomas M. |journal=Protein Science |volume=20 |pmid=21080425 |issue=1}}{{cite journal |doi=10.1371/journal.pone.0003822 |title=Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research |year=2008 |editor1-last=Imhof |editor1-first=Axel |last1=Kobayashi |first1=Takuya |last2=Morone |first2=Nobuhiro |last3=Kashiyama |first3=Taku |last4=Oyamada |first4=Hideto |last5=Kurebayashi |first5=Nagomi |last6=Murayama |first6=Takashi |journal=PLOS ONE |volume=3 |issue=12 |pages=e3822 |pmid=19048102 |pmc=2585475|bibcode=2008PLoSO...3.3822K |doi-access=free }} HDGF-protein complexes were purified using a TAP protocol with the SBP-Tag and with the FLAG-tag{{cite journal |doi=10.1016/j.jprot.2011.08.021 |title=Interactome study suggests multiple cellular functions of hepatoma-derived growth factor (HDGF) |year=2011 |last1=Zhao |first1=Jian |last2=Yu |first2=Hongxiu |last3=Lin |first3=Ling |last4=Tu |first4=Jun |last5=Cai |first5=Lili |last6=Chen |first6=Yanmei |last7=Zhong |first7=Fan |last8=Lin |first8=Chengzhao |last9=He |first9=Fuchu |last10=Yang |first10=Pengyuan |journal=Journal of Proteomics |pmid=21907836 |volume=75 |issue=2 |pages=588–602|display-authors=8 }} and Wnt complexes were purified using a TAP protocol with the SBP-Tag and with the [Calmodulin-Tag].{{cite journal |pages=F685–92 |doi=10.1152/ajprenal.00358.2009 |title=Characterization of the kinase activity of a WNK4 protein complex |year=2009 |last1=Ahlstrom |first1=Robert |last2=Yu |first2=Alan S. L. |journal=AJP: Renal Physiology |volume=297 |issue=3 |pmid=19587141 |pmc=2739714}} 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.{{cite journal |pmid=18719405 |year=2008 |last1=Kyriakakis |first1=Phillip P. |last2=Tipping |first2=Marla |last3=Abed |first3=Louka |last4=Veraksa |first4=Alexey |title=Tandem affinity purification in Drosophila: The advantages of the GS-TAP system |volume=2 |issue=4 |pages=229–35 |journal=Fly |url=http://www.landesbioscience.com/journals/fly/abstract.php?id=6669|doi=10.4161/fly.6669 |doi-access=free }}{{cite journal |pages=1013–9 |doi=10.1038/nmeth968 |title=An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells |year=2006 |last1=Bürckstümmer |first1=Tilmann |last2=Bennett |first2=Keiryn L |last3=Preradovic |first3=Adrijana |last4=Schütze |first4=Gregor |last5=Hantschel |first5=Oliver |last6=Superti-Furga |first6=Giulio |last7=Bauch |first7=Angela |journal=Nature Methods |volume=3 |issue=12 |pmid=17060908|s2cid=7069058 }}{{cite journal |doi=10.1038/msb.2008.75 |title=An integrated workflow for charting the human interaction proteome: Insights into the PP2A system |year=2009 |last1=Glatter |first1=Timo |last2=Wepf |first2=Alexander |last3=Aebersold |first3=Ruedi |last4=Gstaiger |first4=Matthias |journal=Molecular Systems Biology |volume=5 |pmid=19156129 |pages=237 |pmc=2644174 |issue=1}} 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.{{cite journal |pages=1487–99 |doi=10.1007/s10529-011-0592-x |title=The tandem affinity purification technology: An overview |year=2011 |last1=Li |first1=Yifeng |journal=Biotechnology Letters |volume=33 |issue=8 |pmid=21424840|s2cid=157683 }}

=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,{{cite book |pages=195–218 |doi=10.1007/978-1-61779-154-3_11 |isbn=978-1-61779-153-6 |chapter=Isolation of Transcription Factor Complexes from Arabidopsis Cell Suspension Cultures by Tandem Affinity Purification |title=Plant Transcription Factors |editor1-first=Ling |editor1-last=Yuan |editor2-first=Sharyn E |editor2-last=Perry |series=Methods in Molecular Biology |year=2011 |last1=Van Leene |first1=Jelle |last2=Eeckhout |first2=Dominique |last3=Persiau |first3=Geert |last4=Van De Slijke |first4=Eveline |last5=Geerinck |first5=Jan |last6=Van Isterdael |first6=Gert |last7=Witters |first7=Erwin |last8=De Jaeger |first8=Geert |volume=754 |issue=4 |pmid=21720954}} for proteins that interact with the Dengue Virus protein DENV-2 NS4A with the SBP-Tag and the Calmodulin Tag.{{cite journal |pages=17021–9 |doi=10.1074/jbc.M109.006239 |title=The Polypyrimidine Tract-binding Protein Is Required for Efficient Dengue Virus Propagation and Associates with the Viral Replication Machinery |year=2009 |first1=Azlinda |last1=Anwar |first2=K. M. |last2=Leong |first3=Mary L. |last3=Ng |first4=Justin J. H. |last4=Chu |first5=Mariano A. |last5=Garcia-Blanco |journal=Journal of Biological Chemistry |volume=284 |issue=25 |pmid=19380576 |pmc=2719340|doi-access=free }} 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,{{cite journal |first1=Corey M. |last1=McCann |first2=Florence M. |last2=Bareyre |first3=Jeff W. |last3=Lichtman |first4=Joshua R. |last4=Sanes |pmid=16018556 |year=2005 |title=Peptide tags for labeling membrane proteins in live cells with multiple fluorophores |volume=38 |issue=6 |pages=945–52 |journal=BioTechniques |doi=10.2144/05386IT02|doi-access=free }} monitoring of the kinetics of the translation of individual proteins in an in vitro translation system,{{cite journal |pages=9326–32 |doi=10.1021/ja9019947 |title=Real-Time Monitoring of Cell-Free Translation on a Quartz-Crystal Microbalance |year=2009 |last1=Takahashi |first1=Shuntaro |last2=Iida |first2=Masaaki |last3=Furusawa |first3=Hiroyuki |last4=Shimizu |first4=Yoshihiro |last5=Ueda |first5=Takuya |last6=Okahata |first6=Yoshio |journal=Journal of the American Chemical Society |volume=131 |issue=26 |pmid=19518055}} 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.{{cite journal |pages=1441–52 |doi=10.1083/jcb.200707050 |pmc=2373506 |title=Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis |year=2007 |last1=Kida |first1=Yuichiro |last2=Morimoto |first2=Fumiko |last3=Sakaguchi |first3=Masao |journal=The Journal of Cell Biology |volume=179 |issue=7 |pmid=18166653}}{{cite journal |pages=2861–6 |doi=10.1074/jbc.M808020200 |title=Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane |year=2008 |last1=Kida |first1=Y. |last2=Morimoto |first2=F. |last3=Sakaguchi |first3=M. |journal=Journal of Biological Chemistry |volume=284 |issue=5 |pmid=19010775|doi-access=free }} Fluorescent streptavidin reagents (e.g. streptavidin-HRP) can be used to visualize the SBP-tag by immunoblotting of SDS-PAGE.{{cite journal |pages=379–90 |doi=10.1042/BJ20081318 |title=Structural basis and specificity of human otubain 1-mediated deubiquitination |year=2009 |last1=Edelmann |first1=Mariola J. |last2=Iphöfer |first2=Alexander |last3=Akutsu |first3=Masato |last4=Altun |first4=Mikael |last5=Di Gleria |first5=Katalin |last6=Kramer |first6=Holger B. |last7=Fiebiger |first7=Edda |last8=Dhe-Paganon |first8=Sirano |last9=Kessler |first9=Benedikt M. |journal=Biochemical Journal |volume=418 |issue=2 |pmid=18954305 |url=https://hal.science/hal-00479060/file/PEER_stage2_10.1042%252FBJ20081318.pdf }}{{cite journal |pages=45–51 |doi=10.1016/j.febslet.2006.11.075 |title=MARCH-IX mediates ubiquitination and downregulation of ICAM-1 |year=2007 |last1=Hoer |first1=Simon |last2=Smith |first2=Lorraine |last3=Lehner |first3=Paul J. |journal=FEBS Letters |volume=581 |pmid=17174307 |issue=1|s2cid=22461058 |doi-access= }} Additionally, antibodies to the SBP-tag are available commercially.{{citation needed|date=November 2011}}

=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.{{cite journal |pages=1321–6 |doi=10.1007/s00216-006-0794-6 |title=Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip |year=2006 |last1=Li |first1=Yong-Jin |last2=Bi |first2=Li-Jun |last3=Zhang |first3=Xian-En |last4=Zhou |first4=Ya-Feng |last5=Zhang |first5=Ji-Bin |last6=Chen |first6=Yuan-Yuan |last7=Li |first7=Wei |last8=Zhang |first8=Zhi-Ping |journal=Analytical and Bioanalytical Chemistry |volume=386 |issue=5 |pmid=17006676|s2cid=6074268 }} SPR has also been used to compare the SBP-Tag with other streptavidin-binding peptides such as Strep-tag.{{cite journal |pages=435–9 |doi=10.1016/j.jbbm.2006.10.006 |title=Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides |year=2007 |last1=Huang |first1=Xu |last2=Zhang |first2=Xian-En |last3=Zhou |first3=Ya-Feng |last4=Zhang |first4=Zhi-Ping |last5=Cass |first5=Anthony E. G. |journal=Journal of Biochemical and Biophysical Methods |volume=70 |issue=3 |pmid=17156847}}