Strep-tag
Encyclopedia
The Strep-tag® system is a method which allows the purification and detection of protein
s by affinity chromatography
. The Strep-tag is a synthetic peptide
consisting of eight amino acids (Trp
-Ser
-His
-Pro
-Gln
-Phe
-Glu
-Lys
). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin
and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag elutes under gentle, physiological conditions it is especially suited for generation of functional proteins.
is a tetrameric protein expressed in Streptomyces avidinii. Because of its high affinity for the vitamin d-biotin
, Streptavidin is commonly used in the fields of molecular biology
and biotechnology
. The Strep-tag was originally selected from a genetic library to specifically bind to a proteolytically truncated "core" version of streptavidin. Over the years, the Strep-tag was systemically optimized, to permit a greater flexibility in the choice of attachment site. Further, its interaction partner, Streptavidin was also optimized to increase peptide-binding capacity, which resulted in the development of Strep-Tactin. The so called Strep-tag system, consisting of Strep-tag and Strep-Tactin, has proven particularly useful for the functional isolation and analysis of protein complexes in proteome
research.
), the Strep-tag can be easily fused to recombinant
proteins during subcloning of its cDNA or gene
. For its expression various vectors for various host organisms (E. coli
, yeast
, insect
, and mammalian cells) are available. A particular benefit of the Strep-tag is its rather small size and the fact that it is biochemically almost inert
. Therefore, protein folding or secretion is not influenced and usually it does not interfere with protein function. Strep-tag is especially suited for analysis of functional proteins, because the purification procedure can be kept under physiological conditions. This does not only allow the isolation of sensitive proteins in a native state, but it is also possible to purify intact protein complexes, even if just one subunit carries the tag.
In the first step of the Strep-tag purification cycle, the cell lysate containing Strep-tag fusion protein is applied to a column with immobilized Strep-Tactin (step 1). After the tagged protein has specifically bound to Strep-Tactin, a short washing step with a physiological buffer
(e.g. PBS) removes all other host proteins (step 2). This is due to its extraordinary low tendency to bind proteins non specifically. Then, the purified Strep-tag fusion protein is gently eluted with a low concentration of d-desthiobiotin, which specifically competes for the biotin binding pocket (step 3). To regenerate the column, d-desthiobiotin is removed by application of a HABA containing solution (a yellow azo dye). The removal of d-desthiobiotin is indicated by a color change from yellow-orange to red (step 4+5).
Finally, the HABA solution is washed out with a small volume of running buffer, thus making the column ready to use for the next purification run.
(HRP) , alkaline phosphatase
(AP)) or fluorescence (e.g. green fluorescent protein
(GFP)) marker. If high purity of is required, the lysate can be purified by first using Strep-tactin and then perform a second run using antibodies against Strep-tag. This reduces the contamination with unspecific bound proteins, which might occur in some rare scenarios.
Following assays can be conducted using the Strep-tag detection system:
Because the Strep-tag is capable of isolating protein complexes, strategies for
the study of protein-protein interactions can also be conducted. Another option is
the immobilization of Strep-tag proteins with a specific high affinity antibody on microplates or biochips.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s by affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
. The Strep-tag is a synthetic peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
consisting of eight amino acids (Trp
TRP
TRP may refer to:* Trooper, Canadian military abbreviation* Tryptophan, an amino acid* The transient receptor potential channel* Time Reading Program, a book sales club run by Time Life Books in the 1960's* Thomas Ruggles Pynchon, American writer...
-Ser
Ser
Ser is a gender-neutral form of Sir that has been used in the Dragon Age video game series by BioWare, in Greg Bear's The Way novels, and in A Song of Ice and Fire by George R R Martin...
-His
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
-Pro
PRO
PRO may refer to:* Professional, abbreviation for either a person in a profession or in sports* .pro, a sponsored generic top-level domain* Pro Theme, an Apple Macintosh GUI* Pro Hart , Australian painter nicknamed "Pro" for "professor"-PRO:...
-Gln
GLN
GLN or GLn may refer to:* Glutamine, the most abundant naturally occurring amino acid in the human body* Global Location Number, a thirteen digit number used to identify parties and physical locations...
-Phe
PHE
PHE may refer to:* Population Health and Environment , an approach to development that integrates health or family planning with conservation efforts* Paramount Home Entertainment* BitTorrent protocol encryption...
-Glu
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
-Lys
Lys
Lys may refer to any of the following:Places*The Lys or Leie, a river in France and Belgium*Lys , a stream of Aosta Valley in Italy*Lys, Pyrénées-Atlantiques, a commune in the Pyrénées-Atlantiques department in France...
). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered 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 can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag elutes under gentle, physiological conditions it is especially suited for generation of functional proteins.
Development and biochemistry of the Strep-tag
StreptavidinStreptavidin
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...
is a tetrameric protein expressed in Streptomyces avidinii. Because of its high affinity for the vitamin d-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...
, Streptavidin is commonly used in the fields of molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
and biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
. The Strep-tag was originally selected from a genetic library to specifically bind to a proteolytically truncated "core" version of streptavidin. Over the years, the Strep-tag was systemically optimized, to permit a greater flexibility in the choice of attachment site. Further, its interaction partner, Streptavidin was also optimized to increase peptide-binding capacity, which resulted in the development of Strep-Tactin. The so called Strep-tag system, consisting of Strep-tag and Strep-Tactin, has proven particularly useful for the functional isolation and analysis of protein complexes in proteome
Proteome
The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. The term is a portmanteau of proteins and genome.The term has been...
research.
The Strep-tag principle
Just like other short-affinity tags (His-tag, FLAG-tagFLAG-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...
), the Strep-tag can be easily fused to recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
proteins during subcloning of its cDNA or gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
. For its expression various vectors for various host organisms (E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
, yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
, insect
Insect
Insects are a class of living creatures within the arthropods that have a chitinous exoskeleton, a three-part body , three pairs of jointed legs, compound eyes, and two antennae...
, and mammalian cells) are available. A particular benefit of the Strep-tag is its rather small size and the fact that it is biochemically almost inert
Inert
-Chemistry:In chemistry, the term inert is used to describe a substance that is not chemically reactive.The noble gases were previously known as inert gases because of their perceived lack of participation in any chemical reactions...
. Therefore, protein folding or secretion is not influenced and usually it does not interfere with protein function. Strep-tag is especially suited for analysis of functional proteins, because the purification procedure can be kept under physiological conditions. This does not only allow the isolation of sensitive proteins in a native state, but it is also possible to purify intact protein complexes, even if just one subunit carries the tag.
In the first step of the Strep-tag purification cycle, the cell lysate containing Strep-tag fusion protein is applied to a column with immobilized Strep-Tactin (step 1). After the tagged protein has specifically bound to Strep-Tactin, a short washing step with a physiological buffer
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
(e.g. PBS) removes all other host proteins (step 2). This is due to its extraordinary low tendency to bind proteins non specifically. Then, the purified Strep-tag fusion protein is gently eluted with a low concentration of d-desthiobiotin, which specifically competes for the biotin binding pocket (step 3). To regenerate the column, d-desthiobiotin is removed by application of a HABA containing solution (a yellow azo dye). The removal of d-desthiobiotin is indicated by a color change from yellow-orange to red (step 4+5).
Finally, the HABA solution is washed out with a small volume of running buffer, thus making the column ready to use for the next purification run.
Strep-tag applications
The Strep-tag system offers a highly selective tool to purify proteins under physiological conditions. The proteins obtained are bioactive and display a very high purity (above 95%). Also, the Strep-tag system can be used for protein detection in various assays. Depending on the experimental circumstances, Strep-tag antibodies or Strep-tactin, with an enzymatic (e.g.horseradish peroxidaseHorseradish peroxidase
The enzyme horseradish peroxidase , found in horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.-Applications:...
(HRP) , alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
(AP)) or fluorescence (e.g. green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP)) marker. If high purity of is required, the lysate can be purified by first using Strep-tactin and then perform a second run using antibodies against Strep-tag. This reduces the contamination with unspecific bound proteins, which might occur in some rare scenarios.
Following assays can be conducted using the Strep-tag detection system:
- one-step affinity purification
- Protein:protein interaction studies
- Colony blot, dot blotDot blotright|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate Dot-blot filters.A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods...
, Western blotWestern blotThe western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
and ELISAELISAEnzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an... - Screening for positive expression clones
- ImmunocytochemistryImmunocytochemistryImmunocytochemistry is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. These bound antibodies can then be detected using several different methods. ICC allows researchers to evaluate whether or not cells in a...
and ImmunohistochemistryImmunohistochemistryImmunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and... - Protein localization and targeting studies
Because the Strep-tag is capable of isolating protein complexes, strategies for
the study of protein-protein interactions can also be conducted. Another option is
the immobilization of Strep-tag proteins with a specific high affinity antibody on microplates or biochips.