The Experts below are selected from a list of 111417 Experts worldwide ranked by ideXlab platform
Mark Howarth - One of the best experts on this subject based on the ideXlab platform.
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Plug-and-play pairing via defined divalent Streptavidins.
Journal of molecular biology, 2013Co-Authors: Michael Fairhead, Denis Krndija, E.d. Lowe, Mark HowarthAbstract:Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of Streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate Streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent Streptavidin to 1.4 A resolution and trans-divalent Streptavidin to 1.6 A resolution, validating the isolation strategy and explaining the behavior of the Dead Streptavidin variant. cis- and trans-divalent Streptavidins retained tetravalent Streptavidin's high thermostability and low off-rate. These defined divalent Streptavidins enabled us to uncover how Streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent Streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent Streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent Streptavidin, for ultra-stable labeling without undesired clustering. These forms of Streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.
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a Streptavidin variant with slower biotin dissociation and increased mechanostability
Nature Methods, 2010Co-Authors: Claire E Chivers, Vincent T Moy, David J. Sherratt, Estelle Crozat, Calvin Chu, Mark HowarthAbstract:Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant Streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced Streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.
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a Streptavidin variant with slower biotin dissociation and increased
2010Co-Authors: Vincent T Moy, David J. Sherratt, Mark HowarthAbstract:Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, dna amplification and nanotechnology. We identified a mutant Streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. ftsK, a motor protein important in chromosome segregation, rapidly displaced Streptavidin from biotinylated dna, whereas traptavidin resisted displacement, indicating the force generated by ftsk translocation.
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Imaging proteins in live mammalian cells with biotin ligase and monovalent Streptavidin
Nature protocols, 2008Co-Authors: Mark Howarth, Alice Y. TingAbstract:This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by Streptavidin conjugates. This protocol describes the optimal use of BirA and Streptavidin for site-specific labeling and also how to produce BirA and monovalent Streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of Streptavidin's applications. Monovalent Streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type Streptavidin. Site-specific biotinylation and Streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent Streptavidin takes 8 d.
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A monovalent Streptavidin with a single femtomolar biotin binding site
Nature Methods, 2006Co-Authors: Mark Howarth, Daniel J.-f. Chinnapen, Kimberly Gerrow, Pieter C. Dorrestein, Melanie R. Grandy, Neil L. Kelleher, Alaa El-husseini, Alice Y. TingAbstract:Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 104-fold because part of the binding site comes from a neighboring subunit. Here we engineered a Streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type Streptavidin. In denaturant, we mixed a Streptavidin variant containing three mutations that block biotin binding with wild-type Streptavidin in a 3:1 ratio. Then we generated monovalent Streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent Streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type Streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent Streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.
Takeshi Sano - One of the best experts on this subject based on the ideXlab platform.
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A Streptavidin mutant useful for directed immobilization on solid surfaces.
Bioconjugate chemistry, 2001Co-Authors: Gabriel O. Reznik, Sandor Vajda, Charles R. Cantor, Takeshi SanoAbstract:A Streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of Streptavidin on solid surfaces. This Streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of Streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This Streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized Streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This Streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of Streptavidin-based solid-phase assay systems.
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[19] Streptavidin-containing chimeric proteins: design and production
Methods in enzymology, 2000Co-Authors: Takeshi Sano, Charles R. CantorAbstract:Publisher Summary This chapter presents the design and production of Streptavidin-containing chimeric proteins. It also describes the general protocols for expressing Streptavidin-containing chimeras in E. coli by using the bacteriophage T7 expression system and procedures for purifying and characterizing expressed Streptavidin-containing chimeric proteins. Streptavidin is a tetrameric protein produced by the bacterium Streptomyces avidinii , and it has an estimated biotin-binding affinity ( K d ) at 10 –14 M , similar to that of avidin. Its great similarity to avidin, including the biotin-binding and structural characteristics, resulted in the naming of this protein as the streptomyces equivalent of avidin. The tetrameric structure is essential for its extremely tight biotin-binding affinity because intersubunit contacts to biotin, made by an adjacent subunit through a subunit-subunit interface, have a significant contribution to the biotin-binding site. The three-dimensional structured Streptavidin suggests that, without significant modifications, Streptavidin would not be able to form a stable, functional molecule in a dimeric or monomeric form, although a dimeric Streptavidin with reduced biotin-binding affinity and stability has already been produced. When a partner protein is fused to Streptavidin, the resulting Streptavidin-containing chimeric protein forms a tetramer via its Streptavidin moiety, making the fused partner protein also tetrameric.
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Genetic engineering of Streptavidin, a versatile affinity tag
Journal of chromatography. B Biomedical sciences and applications, 1998Co-Authors: Takeshi Sano, Sandor Vajda, Charles R. CantorAbstract:Streptavidin, a tetrameric protein produced by Streptomyces avidinii, has been used as a useful, versatile affinity tag in a variety of biological applications. The efficacy of Streptavidin is derived from its extremely high binding affinity for the vitamin biotin. For the last several years, we have used genetic engineering as a primary means to enhance the properties of Streptavidin and to expand the application of Streptavidin as an affinity tag. In this review, we describe several genetically engineered Streptavidin variants, which include a Streptavidin with a reduced biotin-binding affinity, a dimeric Streptavidin, and a fusion protein between Streptavidin and protein A, along with their potential applications in biological science.
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Properties and Applications of Genetically Engineered Streptavidins
Scientific and Clinical Applications of Magnetic Carriers, 1997Co-Authors: Takeshi Sano, Gabriel O. Reznik, Sandor Vajda, Charles R. Cantor, Cassandra L. SmithAbstract:The wide application of magnetic bead technology has been greatly facilitated by the use of Streptavidin (avidin)-biotin systems, with which a variety of biological materials can be tightly immobilized on solid surfaces. The Streptavidin gene was cloned from Streptomyces avidinii, and it was expressed at high levels in Escherichia coli despite potent antibacterial action of the gene product. The DNA sequence has been modified to provide Streptavidin with a number of basic variations in size, stability, and biotin-binding characteristics, and to construct a system that allows the design of multi-functional proteins by making novel gene fusions with the Streptavidin gene. These genetically engineered Streptavidins are extremely useful for a variety of biotechnological applications, including those with magnetic beads.
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Streptavidins with intersubunit crosslinks have enhanced stability
Nature biotechnology, 1996Co-Authors: Gabriel O. Reznik, Sandor Vajda, Charles R. Cantor, Cassandra L. Smith, Takeshi SanoAbstract:Natural tetrameric Streptavidin has two subunit interfaces; one is a strong interface between subunits in a tightly associated dimer, and the other is a weak interface between a pair of such dimers (dimer-dimer interface). To test whether strengthening the weak dimer-dimer interface could provide Streptavidin with additional structural stability, covalent crosslinks were introduced between adjacent subunits through the dimer-dimer interface. Specific crosslinking sites were designed by site-directed mutations of His-127 residues that are in close proximity in natural Streptavidin. The first and second Streptavidin constructs have a disulfide bond and an irreversible covalent bond, respectively, between two Cys-127 residues across the dimer-dimer interface. The third variant is a hybrid tetramer consisting of two different Streptavidin species, one having lysine and the other aspartic acid at position 127, which are covalently crosslinked. All Streptavidin constructs with intersubunit crosslinks showed higher biotin-binding ability than natural core Streptavidin after heat treatment. All of these crosslinked Streptavidins retained bound biotin more stably than natural core Streptavidin in guanidine hydrochloride at very acidic pH. These results suggest that the introduction of covalent bonds across the dimer-dimer interface enhances the overall stability of Streptavidin.
Charles R. Cantor - One of the best experts on this subject based on the ideXlab platform.
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A Streptavidin mutant useful for directed immobilization on solid surfaces.
Bioconjugate chemistry, 2001Co-Authors: Gabriel O. Reznik, Sandor Vajda, Charles R. Cantor, Takeshi SanoAbstract:A Streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of Streptavidin on solid surfaces. This Streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of Streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This Streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized Streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This Streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of Streptavidin-based solid-phase assay systems.
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[19] Streptavidin-containing chimeric proteins: design and production
Methods in enzymology, 2000Co-Authors: Takeshi Sano, Charles R. CantorAbstract:Publisher Summary This chapter presents the design and production of Streptavidin-containing chimeric proteins. It also describes the general protocols for expressing Streptavidin-containing chimeras in E. coli by using the bacteriophage T7 expression system and procedures for purifying and characterizing expressed Streptavidin-containing chimeric proteins. Streptavidin is a tetrameric protein produced by the bacterium Streptomyces avidinii , and it has an estimated biotin-binding affinity ( K d ) at 10 –14 M , similar to that of avidin. Its great similarity to avidin, including the biotin-binding and structural characteristics, resulted in the naming of this protein as the streptomyces equivalent of avidin. The tetrameric structure is essential for its extremely tight biotin-binding affinity because intersubunit contacts to biotin, made by an adjacent subunit through a subunit-subunit interface, have a significant contribution to the biotin-binding site. The three-dimensional structured Streptavidin suggests that, without significant modifications, Streptavidin would not be able to form a stable, functional molecule in a dimeric or monomeric form, although a dimeric Streptavidin with reduced biotin-binding affinity and stability has already been produced. When a partner protein is fused to Streptavidin, the resulting Streptavidin-containing chimeric protein forms a tetramer via its Streptavidin moiety, making the fused partner protein also tetrameric.
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Genetic engineering of Streptavidin, a versatile affinity tag
Journal of chromatography. B Biomedical sciences and applications, 1998Co-Authors: Takeshi Sano, Sandor Vajda, Charles R. CantorAbstract:Streptavidin, a tetrameric protein produced by Streptomyces avidinii, has been used as a useful, versatile affinity tag in a variety of biological applications. The efficacy of Streptavidin is derived from its extremely high binding affinity for the vitamin biotin. For the last several years, we have used genetic engineering as a primary means to enhance the properties of Streptavidin and to expand the application of Streptavidin as an affinity tag. In this review, we describe several genetically engineered Streptavidin variants, which include a Streptavidin with a reduced biotin-binding affinity, a dimeric Streptavidin, and a fusion protein between Streptavidin and protein A, along with their potential applications in biological science.
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Properties and Applications of Genetically Engineered Streptavidins
Scientific and Clinical Applications of Magnetic Carriers, 1997Co-Authors: Takeshi Sano, Gabriel O. Reznik, Sandor Vajda, Charles R. Cantor, Cassandra L. SmithAbstract:The wide application of magnetic bead technology has been greatly facilitated by the use of Streptavidin (avidin)-biotin systems, with which a variety of biological materials can be tightly immobilized on solid surfaces. The Streptavidin gene was cloned from Streptomyces avidinii, and it was expressed at high levels in Escherichia coli despite potent antibacterial action of the gene product. The DNA sequence has been modified to provide Streptavidin with a number of basic variations in size, stability, and biotin-binding characteristics, and to construct a system that allows the design of multi-functional proteins by making novel gene fusions with the Streptavidin gene. These genetically engineered Streptavidins are extremely useful for a variety of biotechnological applications, including those with magnetic beads.
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Streptavidins with intersubunit crosslinks have enhanced stability
Nature biotechnology, 1996Co-Authors: Gabriel O. Reznik, Sandor Vajda, Charles R. Cantor, Cassandra L. Smith, Takeshi SanoAbstract:Natural tetrameric Streptavidin has two subunit interfaces; one is a strong interface between subunits in a tightly associated dimer, and the other is a weak interface between a pair of such dimers (dimer-dimer interface). To test whether strengthening the weak dimer-dimer interface could provide Streptavidin with additional structural stability, covalent crosslinks were introduced between adjacent subunits through the dimer-dimer interface. Specific crosslinking sites were designed by site-directed mutations of His-127 residues that are in close proximity in natural Streptavidin. The first and second Streptavidin constructs have a disulfide bond and an irreversible covalent bond, respectively, between two Cys-127 residues across the dimer-dimer interface. The third variant is a hybrid tetramer consisting of two different Streptavidin species, one having lysine and the other aspartic acid at position 127, which are covalently crosslinked. All Streptavidin constructs with intersubunit crosslinks showed higher biotin-binding ability than natural core Streptavidin after heat treatment. All of these crosslinked Streptavidins retained bound biotin more stably than natural core Streptavidin in guanidine hydrochloride at very acidic pH. These results suggest that the introduction of covalent bonds across the dimer-dimer interface enhances the overall stability of Streptavidin.
Vincent T Moy - One of the best experts on this subject based on the ideXlab platform.
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a Streptavidin variant with slower biotin dissociation and increased mechanostability
Nature Methods, 2010Co-Authors: Claire E Chivers, Vincent T Moy, David J. Sherratt, Estelle Crozat, Calvin Chu, Mark HowarthAbstract:Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant Streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced Streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.
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a Streptavidin variant with slower biotin dissociation and increased
2010Co-Authors: Vincent T Moy, David J. Sherratt, Mark HowarthAbstract:Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, dna amplification and nanotechnology. We identified a mutant Streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. ftsK, a motor protein important in chromosome segregation, rapidly displaced Streptavidin from biotinylated dna, whereas traptavidin resisted displacement, indicating the force generated by ftsk translocation.
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energy landscape of Streptavidin biotin complexes measured by atomic force microscopy
Biochemistry, 2000Co-Authors: Chunbo Yuan, Aileen Chen, Pamela Kolb, Vincent T MoyAbstract:The dissociation of ligand and receptor involves multiple transitions between intermediate states formed during the unbinding process. In this paper, we explored the energy landscape of the Streptavidin−biotin interaction by using the atomic force microscope (AFM) to measure the unbinding dynamics of individual ligand−receptor complexes. The rupture force of the Streptavidin−biotin bond increased more than 2-fold over a range of loading rates between 100 and 5000 pN/s. Moreover, the force measurements showed two regimes of loading in the Streptavidin−biotin force spectrum, revealing the presence of two activation barriers in the unbinding process. Parallel experiments carried out with a Streptavidin mutant (W120F) were used to investigate the molecular determinants of the activation barriers. From these experiments, we attributed the outer activation barrier in the energy landscape to the molecular interaction of the ‘3-4' loop of Streptavidin that closes behind biotin.
Sheldon Park - One of the best experts on this subject based on the ideXlab platform.
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Streptavidin–biotin technology: improvements and innovations in chemical and biological applications
Applied Microbiology and Biotechnology, 2013Co-Authors: Christopher M. Dundas, Daniel Demonte, Sheldon ParkAbstract:Streptavidin and its homologs (together referred to as Streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the Streptavidin–biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new Streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications. There have been notable developments in the areas of catalysis, cell biology, and proteomics in addition to continued applications in the more established areas of detection, labeling and drug delivery. This review summarizes recent advances in Streptavidin engineering and new applications based on the Streptavidin–biotin interaction.
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Streptavidin-biotin technology: improvements and innovations in chemical and biological applications.
Applied microbiology and biotechnology, 2013Co-Authors: Christopher M. Dundas, Daniel Demonte, Sheldon ParkAbstract:Streptavidin and its homologs (together referred to as Streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the Streptavidin–biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new Streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications. There have been notable developments in the areas of catalysis, cell biology, and proteomics in addition to continued applications in the more established areas of detection, labeling and drug delivery. This review summarizes recent advances in Streptavidin engineering and new applications based on the Streptavidin–biotin interaction.
