SBP-tag
Encyclopedia
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.
. 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.
matrices that bind to the more commonly used 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.
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
.
, 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.
by fusing the SBP-Tag to the N-terminal translocation sequence and then halting integration with streptavidin and re-starting 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.
(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
.
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
sequence that may be engineered into recombinant
Recombinant
Recombinant may refer to:* A recombinant organism - an organism that contains a different combination of alleles from either of its parents.* Recombinant DNA - a form of artificial DNA* Recombinant virus - a virus formed by recombining genetic material...
proteins. Recombinant proteins containing the SBP-Tag bind to streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
and this property may be utilized in specific purification, detection or immobilization strategies.
Discovery
The Streptavidin-Binding Peptide was discovered within a library of seven trillion stochastically-generated peptides using the in vitro selection technique of mRNA DisplayMRNA display
mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target. The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage. The complex then binds to...
. Selection was performed by incubating with streptavidin-agarose followed by elution with biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
. The SBP-Tag has been shown to bind streptavidin with an equilibrium dissociation constant
Dissociation constant
In chemistry, biochemistry, and pharmacology, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into...
of 2.5nM and is readily eluted with biotin under native conditions.
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 IMACIMAC
iMac is a line of Apple Macintosh computers.IMAC or Imac may also refer to:*Necmettin Imac , Netherlands footballer*Isochronous media access controller, a method of transferring data that must not be interrupted ....
matrices that bind to the more commonly used 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 CondensinCondensin
Condensins are large protein complexes that play a central role in chromosome assembly and segregation in eukaryotic cells.- Subunit composition :...
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
GFP
The initialism GFP may refer to:* GFP Personal Finance Manager, a multiplatform Open Source Personal Finance Manager software* GFP Ramdir Sena, a militant Hindu nationalist group in Nepal...
fusion proteins and BTK
BTK
BTK may refer to:*Baku-Tbilisi-Kars Railway*Dennis Rader, a serial killer known as the "BTK killer"**"Bind Torture Kill", a song by the band Suffocation on its album Suffocation....
-protein complexes were purified using a TAP protocol with the SBP-Tag and the His-Tag, HDGF
HDGF
Hepatoma-derived growth factor also known as high mobility group protein 1-like 2 is a protein that in humans is encoded by the HDGF gene....
-protein complexes were purified using a TAP protocol with the SBP-Tag and with the FLAG-Tag
FLAG-tag
FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism...
and Wnt
WNT
WNT is a three-letter abbreviation with multiple meanings, as described below:*Windows NT*WNT *Wnt signaling pathway, a complex protein network...
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
Agilent Technologies
Agilent Technologies , or Agilent, is a company that designs and manufactures electronic and bio-analytical measurement instruments and equipment for measurement and evaluation...
, similar products are sold by Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich Corporation , is a life science and high technology company with over 7,600 employees and operations in 40 countries. Its chemical and biochemical products and kits are used in scientific research, biotechnology, pharmaceutical development, the diagnosis of disease, and as key...
.
Proteomics
Screens for biologically relevant protein-protein interactions have been performed using Tandem-Affinity Purification (TAP) with the SBP-Tag and Protein AProtein A
Protein A is a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in...
, for interaction proteomics and transcription factor complexes with the SBP-Tag and Protein G
Protein G
Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing specificities. It is a 65-kDa and a 58 kDa cell surface protein that has found application in purifying antibodies through its binding to the Fc region...
, for proteins that interact with the Dengue Virus
Dengue virus
Dengue virus in one of four serotypes is the cause of dengue fever. It is a mosquito-borne single positive-stranded RNA virus of the family Flaviviridae; genus Flavivirus...
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 reticulumEndoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
by fusing the SBP-Tag to the N-terminal translocation sequence and then halting integration with streptavidin and re-starting 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 resonanceSurface plasmon resonance
The excitation of surface plasmons by light is denoted as a surface plasmon resonance for planar surfaces or localized surface plasmon resonance for nanometer-sized metallic structures....
(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
Strep-tag
The Strep-tag® system is a method which allows the purification and detection of proteins by affinity chromatography. The Strep-tag is a synthetic peptide consisting of eight amino acids...
.