Streptavidin
Encyclopedia
Streptavidin is a 60000 dalton
protein
purified from the bacterium Streptomyces
avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin
(also known as vitamin B7). With a dissociation constant
(Kd) on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride
), detergents (e.g. SDS
, Triton), proteolytic enzymes, and extremes of temperature and pH.
. The N and C termini of the 159 residue full-length protein are processed to give a shorter ‘core’ streptavidin, usually composed of residues 13 - 139; removal of the N and C termini is necessary for the high biotin-binding affinity. The secondary structure of a streptavidin monomer is composed of eight antiparallel β-strands, which fold to give an antiparallel beta barrel
tertiary structure. A biotin
binding-site is located at one end of each β-barrel. Four identical streptavidin monomers (i.e. four identical β-barrels) associate to give streptavidin’s tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from neighbouring subunit. In this way, each subunit contributes to the binding site on the neighbouring subunit, and so the tetramer can also be considered a dimer of functional dimers.
contacts and hydrophobic interactions made to the biotin when in the pocket, which is also thought to account for the high affinity. In particular, the pocket is lined with conserved tryptophan residues. Lastly, biotin binding is accompanied by the stabilisation of a flexible loop connecting B strands 3 and 4 (L3/4), which closes over the bound biotin, acting like a 'lid' over the binding pocket and contributing to the extremely slow biotin dissociation rate.
Most attempts at mutating streptavidin result in a lowered biotin-binding affinity, which is to be expected in such a highly optimised system. However, a recently engineered mutant of streptavidin, named traptavidin, was found to have more than ten-fold slower biotin dissociation, in addition to higher thermal and mechanical stability. This decreased dissociation rate was accompanied by a two-fold decrease in the association rate.
, which is an optimized system for the purification and detection of proteins. Streptavidin is widely used in Western blotting and immunoassays conjugated to some reporter molecule, such as horseradish peroxidase
.
Pretargetted Immunotherapy
This uses streptavidin conjugated to a monoclonal antibody against cancer cell-specific antigens followed by an injection of radiolabelled biotin, to deliver the radiation only to the cancerous cell. Initial hurdles involve saturation of the biotin binding sites on streptavidin with endogenous biotin instead of the injected radiolabelled biotin, and a high degree of radioactive exposure in the kidneys, due to streptavidin’s strong cell adsorptive properties. It is currently thought that this high level of binding to adherent cell types, such as activated platelets and melanomas, is a result of integrin binding mediated through the RYD sequence in streptavidin.
Monomeric streptavidin is a recombinant form of streptavidin with mutations to break the tetramer into a monomer and to enhance the solubility of the resultant isolated subunit. Monomeric streptavidin has an affinity for biotin of 10-7mol/L and so is not ideal for labeling applications but is useful for purification, where reversibility is desirable.
is the other most notable biotin-binding protein. Originally isolated from egg white, avidin only has 30% sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure. It has a higher affinity for biotin (Kd ~ 10-15M) but in contrast to streptavidin, it is glycosylated, positively charged, has pseudo-catalytic activity (it can enhance the alkaline hydrolysis of an ester linkage between biotin and a nitrophenyl group) and has a higher tendency for aggregation. Also, streptavidin is the better biotin-conjugate binder; avidin has a lower binding affinity than streptavidin when biotin is conjugated to another molecule, despite avidin having the higher affinity for free, unconjugated biotin.
Streptavidin has a mildly acidic isoelectric point
(pI) of ~5, but a recombinant
form of streptavidin with a near-neutral pI is also commercially available. Because streptavidin lacks any carbohydrate
modification and has a near-neutral pI, it has the advantage of much lower nonspecific binding than avidin. Deglycosylated avidin (NeutrAvidin)
is more comparable to the size, pI and nonspecific binding of streptavidin.
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
protein
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...
purified from the bacterium Streptomyces
Streptomyces
Streptomyces is the largest genus of Actinobacteria and the type genus of the family Streptomycetaceae. Over 500 species of Streptomyces bacteria have been described. As with the other Actinobacteria, streptomycetes are gram-positive, and have genomes with high guanine and cytosine content...
avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for 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...
(also known as vitamin B7). With a 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...
(Kd) on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride
Guanidinium chloride
Guanidinium chloride or guanidine hydrochloride, usually abbreviated GdmCl and sometimes GndCl or GuHCl, is the hydrochloride salt of guanidine.-Use in protein denaturation:...
), detergents (e.g. SDS
SDS
-Science:* Safety data sheet or material safety data sheet, a form with data regarding the properties of a particular substance* Satellite Data System, a system of United States military communications satellites....
, Triton), proteolytic enzymes, and extremes of temperature and pH.
Structure
The crystal structure of streptavidin with biotin bound was first solved in 1989 by Hendrickson et al. and as of May 2009, there are 134 structures deposited in the Protein Data BankProtein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....
. The N and C termini of the 159 residue full-length protein are processed to give a shorter ‘core’ streptavidin, usually composed of residues 13 - 139; removal of the N and C termini is necessary for the high biotin-binding affinity. The secondary structure of a streptavidin monomer is composed of eight antiparallel β-strands, which fold to give an antiparallel beta barrel
Beta barrel
A beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last.Beta-strands in beta-barrels are typically arranged in an antiparallel fashion...
tertiary structure. A 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...
binding-site is located at one end of each β-barrel. Four identical streptavidin monomers (i.e. four identical β-barrels) associate to give streptavidin’s tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from neighbouring subunit. In this way, each subunit contributes to the binding site on the neighbouring subunit, and so the tetramer can also be considered a dimer of functional dimers.
Origins of the high affinity
The numerous crystal structures of the streptavidin-biotin complex have shed light on the origins of the remarkable affinity. Firstly, there is high shape complementarity between the binding pocket and biotin. Secondly, there is an extensive network of hydrogen bonds formed to biotin when in the binding site. There are eight hydrogen bonds directly made to residues in the binding site (the so called 'first shell' of hydrogen bonding), involving residues Asn23, Tyr43, Ser27, Ser45, Asn49, Ser88, Thr90 and Asp128. There is also a 'second shell' of hydrogen bonding involving residues that interact with the first shell residues. However, the streptavidin-biotin affinity exceeds that which would be predicted from the hydrogen bonding interactions alone, alluding to another mechanism contributing to the high affinity. The biotin-binding pocket is hydrophobic, and there are numerous van der WaalsVan der Waals
-People:* Fransje van der Waals , Dutch medical physician* Johannes Diderik van der Waals , Dutch physicist-Physics:* the Van der Waals force, named after the physicist* the Van der Waals equation, named after the physicist...
contacts and hydrophobic interactions made to the biotin when in the pocket, which is also thought to account for the high affinity. In particular, the pocket is lined with conserved tryptophan residues. Lastly, biotin binding is accompanied by the stabilisation of a flexible loop connecting B strands 3 and 4 (L3/4), which closes over the bound biotin, acting like a 'lid' over the binding pocket and contributing to the extremely slow biotin dissociation rate.
Most attempts at mutating streptavidin result in a lowered biotin-binding affinity, which is to be expected in such a highly optimised system. However, a recently engineered mutant of streptavidin, named traptavidin, was found to have more than ten-fold slower biotin dissociation, in addition to higher thermal and mechanical stability. This decreased dissociation rate was accompanied by a two-fold decrease in the association rate.
Uses in Biotechnology
Among the most common uses are the purification or detection of various biomolecules. The strong streptavidin-biotin bond can be used to attach various biomolecules to one another or onto a solid support. Harsh conditions are needed to break the streptavidin-biotin interaction, which often denatures the protein of interest being purified. However, it has been shown that a short incubation in water above 70°C will reversibly break the interaction without denaturing streptavidin, allowing re-use of the streptavidin solid support. A further application is the so called Strep-tagStrep-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...
, which is an optimized system for the purification and detection of proteins. Streptavidin is widely used in Western blotting and immunoassays conjugated to some reporter molecule, such as horseradish peroxidase
Horseradish 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:...
.
Pretargetted Immunotherapy
This uses streptavidin conjugated to a monoclonal antibody against cancer cell-specific antigens followed by an injection of radiolabelled biotin, to deliver the radiation only to the cancerous cell. Initial hurdles involve saturation of the biotin binding sites on streptavidin with endogenous biotin instead of the injected radiolabelled biotin, and a high degree of radioactive exposure in the kidneys, due to streptavidin’s strong cell adsorptive properties. It is currently thought that this high level of binding to adherent cell types, such as activated platelets and melanomas, is a result of integrin binding mediated through the RYD sequence in streptavidin.
Monovalent and monomeric streptavidin
Streptavidin is a tetramer and each subunit binds biotin with equal affinity. Multivalency is an advantage in some applications, for example where avidity effects improve the ability of molecules attached to streptavidin to detect specific T cells. In other cases, such as the use of streptavidin for imaging specific proteins on cells, multivalency can perturb the function of the protein of interest. Monovalent streptavidin is an engineered recombinant form of streptavidin which is a tetramer but only one of the four binding sites is functional. This single binding site has 10-14 mol/L affinity and cannot cause cross-linking.Monomeric streptavidin is a recombinant form of streptavidin with mutations to break the tetramer into a monomer and to enhance the solubility of the resultant isolated subunit. Monomeric streptavidin has an affinity for biotin of 10-7mol/L and so is not ideal for labeling applications but is useful for purification, where reversibility is desirable.
Comparison to avidin
Streptavidin is not the only protein capable of binding to biotin with high affinity. AvidinAvidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
is the other most notable biotin-binding protein. Originally isolated from egg white, avidin only has 30% sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure. It has a higher affinity for biotin (Kd ~ 10-15M) but in contrast to streptavidin, it is glycosylated, positively charged, has pseudo-catalytic activity (it can enhance the alkaline hydrolysis of an ester linkage between biotin and a nitrophenyl group) and has a higher tendency for aggregation. Also, streptavidin is the better biotin-conjugate binder; avidin has a lower binding affinity than streptavidin when biotin is conjugated to another molecule, despite avidin having the higher affinity for free, unconjugated biotin.
Streptavidin has a mildly acidic isoelectric point
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
(pI) of ~5, but a 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...
form of streptavidin with a near-neutral pI is also commercially available. Because streptavidin lacks any carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...
modification and has a near-neutral pI, it has the advantage of much lower nonspecific binding than avidin. Deglycosylated avidin (NeutrAvidin)
NeutrAvidin
NeutrAvidin protein is a deglycosylated version of avidin, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity...
is more comparable to the size, pI and nonspecific binding of streptavidin.