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Mark Howarth – One of the best experts on this subject based on the ideXlab platform.

  • Plug-and-play pairing via defined divalent Streptavidins.
    Journal of molecular biology, 2013
    Co-Authors: Michael Fairhead, Denis Krndija, E.d. Lowe, Mark Howarth
    Abstract:

    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 chrochromatography 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.

  • a Streptavidin variant with slower biotin dissociation and increased mechanostability
    Nature Methods, 2010
    Co-Authors: Claire E Chivers, Vincent T Moy, David J. Sherratt, Estelle Crozat, Calvin Chu, Mark Howarth
    Abstract:

    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 segrsegregation, rapidly displaced Streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.

  • a Streptavidin variant with slower biotin dissociation and increased
    , 2010
    Co-Authors: Vincent T Moy, David J. Sherratt, Mark Howarth
    Abstract:

    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 segrsegregation, rapidly displaced Streptavidin from biotinylated dna, whereas traptavidin resisted displacement, indicating the force generated by ftsk translocation.

Takeshi Sano – One of the best experts on this subject based on the ideXlab platform.

Charles R. Cantor – One of the best experts on this subject based on the ideXlab platform.

  • A Streptavidin mutant useful for directed immobilization on solid surfaces.
    Bioconjugate chemistry, 2001
    Co-Authors: Gabriel O. Reznik, Sandor Vajda, Charles R. Cantor, Takeshi Sano
    Abstract:

    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.

  • [19] Streptavidin-containing chimeric proteins: design and production
    Methods in enzymology, 2000
    Co-Authors: Takeshi Sano, Charles R. Cantor
    Abstract:

    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.

  • Genetic engineering of Streptavidin, a versatile affinity tag
    Journal of chromatography. B Biomedical sciences and applications, 1998
    Co-Authors: Takeshi Sano, Sandor Vajda, Charles R. Cantor
    Abstract:

    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.

Vincent T Moy – One of the best experts on this subject based on the ideXlab platform.

  • a Streptavidin variant with slower biotin dissociation and increased mechanostability
    Nature Methods, 2010
    Co-Authors: Claire E Chivers, Vincent T Moy, David J. Sherratt, Estelle Crozat, Calvin Chu, Mark Howarth
    Abstract:

    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.

  • a Streptavidin variant with slower biotin dissociation and increased
    , 2010
    Co-Authors: Vincent T Moy, David J. Sherratt, Mark Howarth
    Abstract:

    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.

  • energy landscape of Streptavidin biotin complexes measured by atomic force microscopy
    Biochemistry, 2000
    Co-Authors: Chunbo Yuan, Aileen Chen, Pamela Kolb, Vincent T Moy
    Abstract:

    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 Streptavidinbiotin interaction by using the atomic force microscope (AFM) to measure the unbinding dynamics of individual ligand−receptor complexes. The rupture force of the Streptavidinbiotin 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 Streptavidinbiotin 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.

  • Streptavidin–biotin technology: improvements and innovations in chemical and biological applications
    Applied Microbiology and Biotechnology, 2013
    Co-Authors: Christopher M. Dundas, Daniel Demonte, Sheldon Park
    Abstract:

    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 Streptavidinbiotin 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 Streptavidinbiotin interaction.

  • Streptavidin-biotin technology: improvements and innovations in chemical and biological applications.
    Applied microbiology and biotechnology, 2013
    Co-Authors: Christopher M. Dundas, Daniel Demonte, Sheldon Park
    Abstract:

    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 Streptavidinbiotin 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 Streptavidinbiotin interaction.