Use Of Avidin Research Articles

Fine-tuning protein mechanostability with AI-driven in silico single-molecule approaches

Among the numerous surprising and counter-intuitive biological phenomena explored by scientific research, protein mechanostability is one of the least explored. However, the presence of adaptive mechanical interactions is widespread, from pathogen adhesion to muscle contraction, highlighting their importance to human health. In biotechnology, the broad use of avidin:biotin reveals another example of highly important mechanostable biomolecular interactions. Here, I present a novel modelling approach that provides unique understanding of protein mechanostability, and also a platform for fine-tuning mechanical properties of protein complexes.

Electrochemical sensing of biotin-avidin interaction on gold electrode modified by silver nanoparticles through covalent co-assembling

Electrochemical sensing of biotin-avidin interaction was investigated on gold electrode modified with 3-mercaptopropanoic acid (MPA) successively co-assembled with cysteamine functionalized silver nanoparticles and biotin or avidin. The stable aqueous colloid of cysteamine functionalized silver nanoparticles was synthesized successfully by well-known citric acid reduction method. Gold electrode was first modified with a monolayer of MPA by self-assembly technique. Then, cysteamine functionalized silver nanoparticles and biotin or avidin was co-assembled on the MPA modified gold electrode through an amide linkage. The stepwise modification process was optimized in terms of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurement in [Fe (CN)6]3−/4− redox system by varying the ratio of functionalized silver nanoparticles and biotin or avidin, viz., 1:2, 1:1, 2:1. Based on obtained results, the electrochemical sensing performances of 1:1 silver nanoparticle and biotin or avidin co-assembled gold electrode was performed by CV and EIS for varying concentration of complementary bio-affinity molecules in the same redox system. Both the CV and EIS measurements were found to have good response by resistive and capacitive changes due to coupling with a complementary biomolecule on co-assembled gold electrode. The use of avidin assembled gold electrode had led to much better response towards varying concentration of biotin over that of the biotin assembled gold electrode towards avidin.

Development of a non-biased, high-throughput ELISA for the rapid evaluation of immunogenicity and cross-reactivity

Traditional ELISA-based protein analysis has been predicated on the assumption that proteins bind randomly to the solid surface of the ELISA plate polymer (polystyrene or polyvinyl chloride). Random adherence to the plate ensures equal access to all faces of the protein, an important consideration when evaluating immunogenicity of polyclonal serum samples as well as when examining the cross-reactivity of immune serum against different antigenic variants of a protein. In this study we demonstrate that the soluble form of the surface lipoprotein transferrin binding protein B (TbpB) from three different bacterial pathogens (Neisseria meningitidis, Actinobacillus pleuropneumoniae, and Mannheimia haemolytica) bind the ELISA plate in a manner that consistently obscures the transferrin binding face of the proteins' N-lobe. In order to develop a non-biased ELISA where all faces of the protein are accessible, the strong interaction between biotin and avidin has been exploited by adding a biotin tag to these proteins during Escherichia coli-based cytoplasmic expression and utilizing streptavidin or neutravidin coated ELISA plates for protein capture and display. The use of avidin coated ELISA plates also allows for rapid purification of biotin-tagged proteins from crude E. coli lysates, removing the requirement of prior affinity purification of each protein to be included in the ELISA-based analyses. In proof of concept experiments we demonstrate the utility of this approach for evaluating immunogenicity and cross-reactivity of serum from mice and pigs immunized with TbpBs from human and porcine pathogens.

A simple method for non-denaturing purification of biotin-tagged proteins through competitive elution with free biotin.

The use of avidin or streptavidin in the purification of biotinylated proteins has been highly restricted due to the harsh and denaturing elution conditions. Here, we use biotinylated bovine serum albumin as a working model to demonstrate a simple and rapid method for biotin-tagged protein purification under non-denaturing conditions. The biotinylated bovine serum albumin is specifically bound to the anti-biotin antibody agarose beads and competitively eluted with free biotin under near-neutral conditions. The optimized elution conditions include using 4 mg/ml biotin (pH 8.5) as the elution buffer and allowing the buffer to incubate with agarose beads for 30min prior to elution. The elution recovery rate is over 85% without apparent protein denaturation. The method is applicable for both immunoprecipitation and column chromatography.

New bioanalytical platform based on the use of avidin for the successful exfoliation of multi-walled carbon nanotubes and the robust anchoring of biomolecules. Application for hydrogen peroxide biosensing

We are reporting an innovative building-block for the development of biosensors based on the non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with avidin (MWCNTs-avidin). In this work, at variance with previous reports, avidin has the double role of simultaneously being the exfoliating agent of MWCNTs and the platform for anchoring different biotinylated biomolecules. The optimum dispersion was obtained by sonicating for 5.0 min 0.50 mgmL−1 MWCNTs with 1.00 mgmL−1 avidin solution prepared in 50:50 v/v ethanol/water. As proof-of-concept, we immobilized biotinylated horseradish peroxidase (b-HRP) at glassy carbon electrodes (GCE) modified with MWCNTs-avidin to develop a hydrogen peroxide biosensor using hydroquinone as redox mediator. Surface plasmon resonance, electrochemical impedance spectroscopy, cyclic voltammetry and amperometry demonstrated that, even after the partial denaturation of avidin due to the drastic conditions used to functionalize the MWCNTs, it preserves the biorecognition properties and efficiently interacts with biotinylated horseradish peroxidase (b-HRP). The analytical characteristics of the resulting hydrogen peroxide biosensor are the following: linear range between 1.0 × 10−6 M and 1.4 × 10−5 M, sensitivity of (1.37 ± 0.04) x 105 μAM−1, detection limit of 24 nM and reproducibility of 2.9%. The sensor was challenged with different samples, a mouthwash solution, human blood serum and milk, with very good performance.

The Sandwich Assay: A Method for Subcellular Visualization of Paracellular Macromolecule Passage in Epithelial Sheets.

To date, the permeability of epithelia to larger solutes (greater than ∼4 Å in diameter) has been analyzed by flux measurements using various tracers that cannot spatially resolve the permeation sites. This unit describes a method for localizing such sites of passage in epithelial sheets with subcellular resolution. The method makes use of avidin as a basolateral capture probe in epithelial monolayers or mucosae to unmask the passage of biotinylated and fluorophore-labeled tracer molecules as they go through the junctional barrier. Once bound to avidin, the tracers are immobilized at the site of a barrier leak. The localization, the distribution, and the extent of passage are eventually evaluated by imaging. The assay detects single leaks and is hence able to spatially resolve rarely occurring changes. It is also modular and flexible to use with various macromolecular tracers, and its sensitivity is adjustable. If designed as a chase experiment, the method allows for analysis of temporal barrier openings. If performed at low temperatures, this assay will block transcellular passage and, combined with global flux measurement, unambiguously determine paracellular passage. © 2018 by John Wiley & Sons, Inc.

Self-assembly and modular functionalization of three-dimensional crystals from oppositely charged proteins.

Multicomponent crystals and nanoparticle superlattices are a powerful approach to integrate different materials into ordered nanostructures. Well-developed, especially DNA-based, methods for their preparation exist, yet most techniques concentrate on molecular and synthetic nanoparticle systems in non-biocompatible environment. Here we describe the self-assembly and characterization of binary solids that consist of crystalline arrays of native biomacromolecules. We electrostatically assembled cowpea chlorotic mottle virus particles and avidin proteins into heterogeneous crystals, where the virus particles adopt a non-close-packed body-centred cubic arrangement held together by avidin. Importantly, the whole preparation process takes place at room temperature in a mild aqueous medium allowing the processing of delicate biological building blocks into ordered structures with lattice constants in the nanometre range. Furthermore, the use of avidin–biotin interaction allows highly selective pre- or post-functionalization of the protein crystals in a modular way with different types of functional units, such as fluorescent dyes, enzymes and plasmonic nanoparticles.

Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein

BackgroundEngineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure.ResultsAvidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ~ 9 μM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between β-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins.ConclusionsThe high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.

Bifunctional Avidin with Covalently Modifiable Ligand Binding Site

The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

Therapeutic Use of Avidin Is Not Hampered by Antiavidin Antibodies in Humans

Hen egg white avidin is increasingly used in the clinic as part of multifactor treatments such as pretargeted radionuclide therapy of cancer or as an antidote of biotinylated drugs. Taking into account that naturally occurring human antiavidin antibodies (HAVA) are common in humans, the present work investigates avidin immunogenicity as part of risk/benefit evaluations. Sera from 139 oncology patients naive to avidin were confirmed to exhibit HAVA with lognormally distributed titers. HAVA were boosted after avidin treatment, with no correlation with the avidin dose or with the basal titer. No antibody-related clinical symptoms were observed in 21 HAVA-positive patients treated with avidin. In mouse models, high mouse antiavidin antibody titers, induced to simulate the worst human condition, neither reduced the biotin uptake of intratissue-injected avidin nor affected the capacity of intravenously injected avidin to clear a biotinylated drug from circulation. In both models the avidin treatment was well tolerated. Results indicate that avidin immunogenicity does not affect its safety and efficacy, thus encouraging its further use in clinical applications.

An improvedin situhybridization method for the detection of fish pathogens

A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.

Directed capture of enzymes and bacteria on bioplastic films.

The development of smart coatings for a variety of uses depends on the ability of the coating material to perform specific functions. We have used water dispersible polyurethane preparations for the immobilization of binding proteins under mild conditions. In these experiments, antibodies against the enzyme beta-galactosidase or the bacterium Escherichia coli were immobilized in polyurethane coatings and then used to effectively capture their cognate antigen. Further, a second, more general, capture protocol was developed which involves the incorporation of the protein avidin in the plastics. This system efficiently captures biotinylated beta-galactosidase. Biotinylated anti-E. coli antibody captured by avidin bioplastics resulted in a nearly 5-fold increase in the number of bound bacteria when compared to blank polyurethane. The use of avidin in a bioplastic allows any biotinylated antibody to be applied to all or part of the surface resulting in a patterning of capture agents on a preformed surface.

Mutation of the important Tyr-33 residue of chicken avidin: functional and structural consequences.

The strong interaction between avidin and biotin is so tight (dissociation constant 10(-15) M) that conditions usually sufficient for protein denaturing fail to dislodge biotin from the avidin-biotin complex. This kind of irreversible binding hinders the use of avidin in applications such as affinity purification or protein immobilization. To address this concern, we have constructed a series of mutants of the strategically positioned Tyr-33 in order to study the role of this residue in biotin binding, and to create avidin variants with more reversible ligand-binding properties. Unexpectedly, an avidin mutant in which Tyr-33 was replaced with phenylalanine (Avm-Y33F) displayed similar biotin-binding characteristics to the native avidin, indicating that the hydrogen bond formed between the hydroxy group of Tyr-33 and the carbonyl oxygen of biotin is not as important for the tight binding of biotin as previously suggested. In terms of the reversibility of biotin binding, Avm-Y33H was the most successful substitution constructed in this study. Interestingly, the binding of this mutant exhibited clear pH-dependence, since at neutral pH it bound to the biotin surface in an irreversible fashion, whereas, at pH 9, 50% of the bound protein could be released with free biotin. Furthermore, although Tyr-33 is located relatively distant from the monomer-monomer interfaces, the mutagenesis of this residue also weakened the quaternary structure of avidin, indicating that the high ligand binding and the high stability of avidin have evolved together and it is difficult to modify one without affecting the other.

Luminescent Quantum Dot-Adaptor Protein-Antibody Conjugates for Use in Fluoroimmunoassays

A method for the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe–ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays is presented. The conjugation strategy employs two routes: 1. Use of an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies, and 2. use of avidin, also electrostatically self-assembled onto the nanocrystal surface, which allows QD conjugation to biotinylated antibodies via avidin–biotin binding scheme. With this approach, the average number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed composition conjugates of the molecular adaptor and a second “inert” two-domain fusion protein that allows the use of affinity chromatography. QD/adaptor-antibody conjugates were successfully employed in fluoroimmunoassays for the detection of small molecule analytes, 2,4,6-trinitrobenzene (TNB) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We also demonstrate the use of QD/avidin-antibody conjugates for fluoroimmunoassays using a model protein system.

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes

The interaction of avidin – a basic protein from hen egg-white – with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.

Immunocytochemical detection of two nuclear proteins within the same neuron using light microscopy

We developed a method of double immunocytochemistry (ICC) that can be used with conventional light microscopy for localizing two different nuclear proteins. The procedure involves two sequential rounds of ICC that both employ the avidin and biotin conjugated enzyme (ABC) amplification method, separated by an Avidin D and biotin blocking step to reduce non-specific avidin–biotin reactions. Round one of ICC employs the use of avidin and biotin conjugated alkaline phosphatase (ABC–AP) and the Vector Red (VR) substrate, which produces a red colorimetric reaction product. The second round of ICC makes use of avidin and biotin conjugated peroxidase (ABC–HRP) and the Vector® SG substrate, which produces a gray colorimetric reaction product. Neuronal nuclei that are double-labeled for both proteins appear red with a gray core. This protocol allows the simultaneous detection of two proteins within the same subcellular compartment of a single neuron, without the need for epifluorescence or scanning confocal laser microscopy. Theme: Cellular and Molecular Biology Term: Staining, tracing, and imaging techniques

Evaluation of a commercial system for the identification of Ornithobacterium rhinotracheale.

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The use of avidin as a probe for the distribution of mitochondrial carboxylases in developing chick retina.

During development, the inner chick retina progresses from an aerobic to an anaerobic metabolic basis because of the lack of a vascular system. To investigate this process further, we have examined the expression and distribution of mitochondrial carboxylases. Because these enzymes use covalently bound biotin as a co-enzyme, we were able to develop a new detection protocol for mitochondria using avidin as a probe for the biotin. Chemiluminescent detection of bound avidin-peroxidase was used to examine a developmental series of extracts of retinas that had been separated by electrophoresis and blotted to nitrocellulose. Avidin-peroxidase, visualized with the sensitive peroxidase substrate True Blue, permitted detection in epoxy-embedded tissue sections. In the extracts, specific bands of approximate molecular weights 130 and 70 kD were found, corresponding to biotinylated subunits of several mitochondrial carboxylases. During development, the intensity of the bands decreases, although at different rates. In tissue sections, 8-day embryonic retinas display reaction product throughout the tissue, with higher local concentrations in the vitread and sclerad regions. During further development, the reaction product becomes segregated into bands at the borders of the plexiform layers. As the photoreceptors mature, stain becomes concentrated in the developing ellipsoids and the sclerad ends of Müller cells.

Recombinant metallothionein-conjugated streptavidin labeled with 188Re and 99mTc.

Consideration is now being given to the use of avidin (or streptavidin) and biotin for radiotherapy of tumor. Accordingly, the goal of this study was to radiolabel a mouse metallothionein-streptavidin fusion protein with 188Re and to compare its properties to those of the same fusion protein radiolabeled with 99mTc. A recombinant metallothionein-streptavidin fusion protein was radiolabeled by transchelation with 99mTc- and 188Re-glucoheptonate. Labeling efficiency, which was not optimized for either radionuclide, was approximately 60% for 99mTc and 20% for 188Re. Radiochemical purity was demonstrated by size exclusion HPLC both by nearly quantitative shifts of the 188Re label to higher molecular weight upon the addition of biotinylated antibody and by the absence of a shift with biotinsaturated 188Re-metallothionein-streptavidin. Stability of the labels in 37 degrees C serum was evaluated by comparing the HPLC radiochromatograms of serum samples both before and after the addition of biotinylated antibody. The 188Re label behaved like 99mTc in that the same peaks were evident, including one prominent peak due to labeled cysteine. Recoveries during HPLC analysis of serum samples showed that oxidation rates to perrhenate and pertechnetate were identical. However, instability to cysteine challenge was greater for 188Re; for example, the loss of label to cysteine after 24 h under one set of conditions was 41% for 188Re and 22% with 99mTc. Analysis by HPLC of liver and kidney homogenates from mice administered the labeled antibodies were qualitatively and, in large measure, quantitatively independent of label. Biodistributions at 5 h in normal mice were statistically identical between the two labels in blood and in most tissues. In conclusion, streptavidin may be radiolabeled with radiorhenium using recombinant mouse metallothionein as a bifunctional chelator, and under one set of labeling conditions at least, 188Re showed similar in vitro and in vivo behavior to that of 99mTc labeled to the same fusion protein.

A novel biotinylated heterobifunctional cross-linking reagent bearing an aromatic diazirine

The synthesis of a p-[(3-trifluoromethyl)diazirine-3-yl]benzoic acid derivative is described as a new carbene generating heterobifunctional cross-linking reagent. The cross-linker carries a biotin moiety in order to make use of avidin—biotin technology for specific manipulation of cross-linked components. To evaluate the ability of this reagent, the inter-subunit cross-linking of egg-white avidin tetramer was investigated. As a typical application of avidin—biotin technology for cross-linking experiments, a chemiluminescent detection method was examined to identify photobiotinylated components. A cross-linked dimeric product with an apparent molecular mass of 38 kDa was clearly visualized by the combined use of a horseradish peroxidase—streptavidin conjugate and a luminol-based chemiluminescent system.