Native Streptavidin Research Articles

Tyrosine bioconjugation with hypervalent iodine.

Hypervalent iodine reagents have recently emerged as powerful tools for late-stage peptide and protein functionalization. Herein we report a tyrosine bioconjugation methodology for the introduction of hypervalent iodine onto biomolecules under physiological conditions. Tyrosine residues were engaged in a selective addition onto the alkynyl bond of ethynylbenziodoxolones (EBX), resulting in stable vinylbenziodoxolones (VBX) bioconjugates. The methodology was successfully applied to peptides and proteins and tolerated all other nucleophilic residues, with the exception of cysteine. The generated VBX were further functionalized by palladium-catalyzed cross-coupling and azide-alkyne cycloaddition reactions. The method could be successfully used to modify bioactive natural products and native streptavidin to enable thiol-mediated cellular uptake.

Intrinsic Förster Resonance Energy Transfer Imaging Technique for Detection of Native Protein in Live Cells

To detect a native target protein and its interaction with drugs in live cells, we developed an intrinsic fluorescence resonance energy transfer (iFRET) imaging technique (iFIT). The iFIT employs a target‐specific fluorescent probe in combination with a deep UV biological microscope. The iFRET utilizes tryptophan residues of the target protein and a cell‐permeable target‐specific probe, which is selectively excited by the intrinsic tryptophan fluorescence (λem = 350 nm), as FRET donors and acceptors, respectively. As the Förster distance between the tryptophan residues and the iFIT probe is about 2 nm, an iFRET signal is generated by specific interaction of the target protein and the selective iFIT probe. A deep UV biological microscope equipped with a quartz objective and two optical filters (288 and 365 nm) on a shutter was constructed to facilitate the detection of the iFRET signal upon binding of the probe to its target, in live cells. We successfully achieved imaging of native streptavidin in live cells with the thus developed iFIT, after simple treatment of live cells with the corresponding iFIT probes.

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Improved surface stability and biotin binding properties of streptavidin coating on polystyrene

The ultimate nature of streptavidin to bind biotin tightly is widely utilized in many solid-phase based applications to provide a universal binding surface for biotinylated molecules. However, the preparation of the streptavidin coatings by passive adsorption may heavily alter the binding properties of native streptavidin and may not result in the best possible capture surface for demanding solid-phase assays. By introducing sulphydryl groups through primary amines in the protein, we have activated and conjugated native streptavidin into larger protein polymers resulting in high local binding density when coated on polystyrene. This thiolated streptavidin formed through chemical modification has improved adsorption properties and biotin binding capability, compared to the native streptavidin. When this thiolated streptavidin is coated on polystyrene, a dense surface is formed, which provides up to 3-fold increase in the biotin binding efficiency and improves the surface stability by minimizing the desorption of the adsorbed protein from the surface during incubation. Furthermore, this high-capacity surface is resistant to harsh chemical treatments, such as denaturing conditions or mild reducing conditions. The improved adsorption properties of the thiolated streptavidin allow the coating process to be performed with shorter incubation times (15 min), still providing enhanced solid-phase properties, compared to a reference streptavidin surface.

Atomic force microscopical and surface plasmon resonance spectroscopical investigation of sub-micrometer metal gratings generated by UV laser-based two-beam interference in Au–Ag bimetallic layers

Metal films containing silver and gold layers having different thicknesses were evaporated on glass substrates. Two-beam interference technique was applied to irradiate the surfaces by the fourth harmonic of a pulsed mode Nd:YAG laser. The atomic force microscopical study showed that surface relief grating having a period of 900 nm corresponding to the interference pattern was developed on the metallic films. The modulation amplitude of the laser-induced gratings was increasable by enhancing the number of laser pulses at constant fluence, and a groove depth commensurable with the film thicknesses was generated at the average fluence of 39.5 mJ/cm 2 on bimetallic layers. The surface structure was more regular, and the modulation amplitude was larger in case of bimetallic films containing thicker gold layers. The threshold fluences of the phase transitions were determined by numerical temperature model calculations for different metal layer compositions, and a good agreement was found between the calculated and experimentally observed threshold values. The division of the metal stripes into droplets and the development of holes were explained by the melting of the entire metal layers and by the vaporization of silver at higher fluences. The angle-dependent surface plasmon resonance spectroscopy realized in Kretschmann arrangement proved that the laser-induced grating formation was accompanied by the change in the optical thickness and by the modification of the structure of the bimetallic films. Broad side wings appeared on the resonance curves caused by grating–coupling in case of appropriate rotation angle and sufficiently large modulation depth of the grating's grooves, according to our calculations. The coupling on deep gratings developed on bimetallic films containing the thinnest gold layer and on monometallic silver films resulted in separated secondary resonance minimum development. The periodic adherence of native streptavidin on the metallic gratings was detected by tapping mode AFM, and based on the shift of the secondary resonance peak.

A TAT–streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells

The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.

Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize.

The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.

Supramolecular DNA-Streptavidin Nanocircles with a Covalently Attached Oligonucleotide Moiety

Covalent hybrid conjugates consisting of streptavidin (STV) and a 24-mer single-stranded DNA oligonucleotide have been used as a starting material for the synthesis of supramolecular nanocircles. For this, the covalent hybrid conjugates were oligomerized by cross-linking with 5′,5′-bis-biotinylated double-stranded DNA (dsDNA) fragments of various length. Heat denaturation of the resulting oligomeric conjugates and subsequent rapid cooling led to the formation of the nanocircles, in which the oligonucleotide-containing STV molecule is coupled with both ends of the circular bis-biotinylated dsDNA fragment. The circular structure of the bioconjugates was established by electrophoretic studies including Ferguson plot analysis as well as by scanning force microscopy (SFM) inspection. The formation process and the stability against degradation by ligand exchange with free D-biotin was compared for the nanocircles obtained from covalent oligonucleotide-STV hybrids and native STV. The former nanocircles revealed a decreased stability with respect to ring opening than the circles obtained from native STV. This suggested that the affinity of the covalent oligonu- cleotide-STV hybrid for binding biotinylated DNA is significantly decreased. Nevertheless, the single-stranded oligonucleotide moiety of the hybrid nanocircles can be used as a molecular handle for further functionalization. For instance, it was used for the selective DNA-directed immobilization at a surface, previously functionalized with complementary capture oligonucleotides. Moreover, we demonstrate that a pair of nanocircles, containing complementary oligonucleotide moieties, can be hybridized to form specific dimers, thereby generating a novel type of supramolecular DNA-protein nanostructures.

Isothiocyanate-trigalactose: application for antibody-targeted delivery of diagnostic and therapeutic agents.

Radiolabeled monoclonal antibodies (MAb) and MAb-streptavidin conjugates exhibit slow blood clearance which impedes radioimmunoimaging and radioimmunotherapy. To control blood clearance and lower background levels, lesion-specific targeting proteins can be modified with galactose derivatives for liver uptake via the hepatocyte galactose receptor. In this study, an isothiocyanate-trigalactose derivative (ITC-Tgal) designed for direct coupling to protein amino groups, was synthesized and characterized. In vitro experimentation demonstrated efficient conjugation of ITC-Tgal to streptavidin (SA) and MAb Fab fragment with a corresponding decrease in protein net charge. In vivo studies were conducted with radiolabeled ITC-Tgal modified and native SA and MAb Fab fragment. ITC-Tgal modified SA and Fab fragment exhibited increased blood clearance with the liver uptake and the rate of blood clearance controlled by the extent of ITC-Tgal modification.

A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway.

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Galactosylated streptavidin for improved clearance of biotinylated intact and F(ab')2 fragments of an anti-tumour antibody.

Persistence of high levels of radiolabelled antibody in the circulation is a major limitation of radioimmunotherapy. Biotinylation of the radiolabelled anti-tumour antibody followed by administration of streptavidin is known to give much improved tumour to blood ratios as the radioantibody is complexed and subsequently cleared via the reticuloendothelial system, although prolonged splenic uptake is a problem. We have investigated the effect on the clearance pattern and tumour localisation of a 125I-labelled biotinylated anti-CEA antibody (A5B7) after administration of a galactosylated form of streptavidin (gal-streptavidin) in nude mice bearing a human colon carcinoma xenograft. Fifteen minutes to 1 h after gal-streptavidin administration the complexes were cleared via the liver alone (as opposed to liver and spleen after native streptavidin). Twenty-four hours after administration of gal-streptavidin, the tumour to blood ratio for biotinylated A5B7 IgG increased from 2.9 to 13.2 and for biotinylated F(ab')2 fragments an increase from 4.9 to 33.2 was achieved. The reduction in tumour accumulation of F(ab')2 24 h after injection of the clearing agent was less than that seen with intact antibody. Injection of asialofetuin inhibited clearance, confirming that removal of the gal-streptavidin-biotinylated antibody complexes from the blood was via the asialoglycoprotein receptor on liver hepatocytes. Therefore, galactosylation of the streptavidin clearing agent allows rapid removal of radiolabelled biotinylated antibodies via the liver asialoglycoprotein receptor, as opposed to the reticuloendothelial system.

The quaternary structure of streptavidin in urea

We report on the interactions of urea and guanidinium salts with streptavidin. Gel filtration chromatography in 0, 4, 6, and 7 M urea indicates that the streptavidin tetramer remains intact in urea. Biotin alters the electrophoretic mobility of streptavidin whether or not 6 M urea is present. The intrinsic fluorescence of streptavidin is increased and blue-shifted in 6 M urea. The fluorescence changes indicate the absence of unfolding. A conformational response to urea is possible, but much of the fluorescence change is due to urea binding as a weak biotin analog (Ka approximately 1.3 M-1). The resistance to structural perturbation by urea reflects the structural stability of streptavidin's anti-parallel beta-barrel motif. Unfolding is sluggish in 6 M guanidinium hydrochloride (half-time, approximately 50 days). After guanidinium thiocyanate unfolding, streptavidin can be refolded, but the unfolding and refolding transitions are centered at different concentrations of perturbant. Slow unfolding, with a 15th power dependence on guanidinium thiocyanate concentration, may be partially responsible for the noncoincidence of the unfolding and refolding processes. Nonequilibrium behavior is also seen in 6 M urea, as native streptavidin does not unfold and guanidinium thiocyanate unfolded streptavidin does not refold. Refolding does occur at lower concentrations of urea. Guanidinium thiocyanate only slowly unfolds the biotin-streptavidin complex. In the presence of biotin, unfolded streptavidin does not refold in 6 M guanidinium thiocyanate or in 6 M urea.

Tissue distribution of avidin and streptavidin injected to mice

Radioionated avidin and streptavidin were characterized for their biodistribution and tissue association in Balb/c mice, in comparison to their interaction with cells in vitro. Binding of avidin to spleen and bone-marrow cells in vitro was up to 20-fold higher than that of streptavidin, but when tested in vivo avidin clearance from blood and tissues was considerably faster than that of streptavidin. Levels of avidin at 24 h after an intravenous injection were below 1% (of the injected dose/mass tissue) in most organs. Non-glycosylated avidin was similar in its biodistribution to native avidin. Native streptavidin exhibited higher and prolonged tissue association with 5-10% levels in lung, liver, spleen, kidney and blood, whereas its truncated form showed low tissue levels (1-3%) but a remarkably high affinity to the kidney (80%). Exogenous biotin did not affect streptavidin distribution in vivo but caused a 2-7-fold increase in the retention of avidin (but not non-glycodylated avidin) in some of the organs.