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Nature's optimization of protein functions is a highly intricate evolutionary process. In addition to optimal tertiary folding, the intramolecular recognition among the monomers that generate higher-order quaternary arrangements is driven by stabilizing interactions that have a pivotal role for ideal activity. Homotetrameric avidin and streptavidin are regularly utilized in many applications, whereby their ultra-high affinity toward biotin is dependent on their quaternary arrangements. In recent years, a new subfamily of avidins was discovered that comprises homodimers rather than tetramers, in which the high affinity toward biotin is maintained. Intriguingly, several of the respective dimers have been shown to assemble into higher-order cylindrical hexamers or octamers that dissociate into dimers upon biotin binding. Here, we present wilavidin, a newly discovered member of the dimeric subfamily, forming hexamers in the apo form, which are uniquely maintained upon biotin binding with six high-affinity binding sites. Removal of the short C-terminal segment of wilavidin resulted in the presence of the dimer only, thus emphasizing the role of this segment in stabilizing the hexamer. Utilization of a hexavalent biotin-binding form of avidin would be beneficial for expanding the biotechnological toolbox. Additionally, this unique family of dimeric avidins and their propensity to oligomerize to hexamers or octamers can serve as a basis for protein oligomerization and intermonomeric recognition as well as cumulative interactions that determine molecular assemblies.

As molecular electronic components, proteins are distinguished by a remarkably long electronic decay length (~10 nm) together with high contact resistance and extreme sensitivity to the chemical details of the contact. As a consequence, the conductance of even a large bioelectronic assembly is largely controlled by the conductance of the contacts. Streptavidin is a versatile linker-protein that can tether together biotinylated electrodes and biotinylated proteins, but with an ambiguity about the contact geometry that arises from its four possible binding sites for biotin. Here, we use engineered streptavidin tetramers, selected to contain a defined ratio of active monomers to ‘dead’ monomers so as to define the biotin binding sites. We find a strong dependence of conductance on the separation of the biotin molecules, consistent with a short-range tunneling interaction within the streptavidin, and in contrast to the long-range transport observed inside larger proteins. Hexaglutamate tails label the active monomers and the additional negative charge enhances conductance significantly. This effect is quantitatively accounted for by an electronic resonance in the protein conductance.

Background Avidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptavidin from Streptomyces avidinii, a growing number of avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for avidin encoding genes among bacteria using available databases and classified potential avidins according to taxonomy and the ecological niches utilized by host bacteria. Results Numerous avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding avidins were found. The living strategies of bacteria hosting avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria. Conclusions Bacterial avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity.

The structural analysis of mature streptavidin C1 from S. cinnamonensis is reported., The members of the avidin protein family are well known for their high affinity towards d-biotin and their structural stability. These properties make avidins a valuable tool for various biotechnological applications. In the present study, two avidin-like biotin-binding proteins (named streptavidin C1 and C2) from Streptomyces cinnamonensis were newly identified while exploring antifungal proteins against Fusarium oxysporum f. sp. cucumerinum. Streptavidin C1 reveals a low correlation (a sequence identity of approximately 64%) with all known streptavidins, whereas streptavidin C2 shares a sequence identity of approximately 94% with other streptavidins. Here, the crystal structures of streptavidin C1 in the mature form and in complex with biotin at 2.1 and 2.5 Å resolution, respectively, were assessed. The overall structures present similar tetrameric features with D 2 symmetry to other (strept)avidin structures. Interestingly, the long C-terminal region comprises a short α-helix (C-Lid; residues 169–179) and an extension C-terminal peptide (ECP; residues 180–191) which stretches into the biotin-binding sites of the same monomer. This ECP sequence (–180VTSANPPAS188–) is a newly defined biotin-binding site, which reduces the ability to bind to (strept)avidin family proteins. The novel streptavidin C1 could help in the development of an engineered tetrameric streptavidin with reduced biotin-binding capacity as well as other biomaterial tools.

An entropically-driven binding interaction at a certain temperature may change to an enthalpically-driven process at another temperature, depending on the polarization state of the groups that are involved in binding. The streptavidin-biotin complex has been extensively studied across biological, medical, chemical and material science fields using various techniques, however, not much has been reported on this interaction across a broad temperature range, between 2 °C and 40 °C using biophysical techniques. In this study, we determined how the forces involved in the streptavidin-biotin complex formation are affected by the reaction temperature using the Affinity ITC (TA Instruments). We observed that this complex formation is a spontaneous binding process, indicated by a negative Gibbs energy (ΔG) at all temperatures tested. The observed negative heat capacity (ΔCp) ∼ −459.9 cal/mol K highlights the polar solvation of the interaction that corresponds to a decreasing enthalpy (more negative) (ΔH) with increasing reaction temperature. The stoichiometry (n) of 0.98 was estimated at 25 °C. An increase in reaction temperature resulted in an almost two-fold increase or more in n, notably from 1.59 to 3.41 between 30 °C and 40 °C. Whereas, at lower reaction temperatures, 2 °C to 10 °C, higher molar binding ratios were recorded, i.e. 2.74 to 5.76. We report an enthalpically-driven interaction between 30 °C and 40 °C whereas, an entropically-favourable interaction is observed at lower temperatures, suggestive of an interaction dominated by nonpolar interactions at lower temperatures and polar interactions at higher temperatures. Consequently, alterations in the polarisation state of streptavidin result in moderate binding affinity of biotin to streptavidin at higher reaction temperatures, KD 10−4 ≤ 10−5 M.

Background Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists. Results We report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples. Conclusion The assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.

Detection of protein-binding analytes is important for many applications. Currently, various instrument-based techniques are used for detecting protein-binding analytes. However, such techniques have several limitations including high cost and time-consuming sample processing. In order to overcome these limitations, we developed a sensitive competition assay for the detection of protein-binding analytes using recombinant endospores as a sensing element. The method is based on the competition between the biotin, the model analyte, and a biotin-magnetic bead complex to bind the recombinant spores containing the biotin binding region of streptavidin. After magnetic attraction, the residual spores in the suspension are spread on plates to form colonies which are used to count the amount of the residual spores; the higher the residual ratio of spores, the more biotin in the samples. The linear range was from 150 zmol to 1.5 fmol and the limit of detection of the assay was 150 zmol. The assay proposed herein is sensitive and does not require any expensive equipment. It is suitable for qualitative or semi-quantitative analysis such as screening tests for the detection of toxic chemicals.

In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin-biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin-biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin-biotin biotechnology in cellular uptake.

Accurate and rapid quantification of foodborne pathogens is of great significance for food safety and human health. In this work, we have successfully constructed a fluorescence quenching collapsar probe (FQCP) on the basis of a conventional aptamer-encoded molecular beacon (AEMB) and applied it for the detection of Salmonella. In structure, the FQCP is assembled by AEMBs in fours via specific streptavidin and biotin binding. Such a simple format makes the FQCP cofunctionalized with short- and long-range fluorescence resonance energy transfer (FRET) effects, thereby leading to a significantly suppressed inherent background fluorescence that is much lower than that of the conventional AEMB. Moreover, the FQCP exhibits superior biostability because of the blocking of its 3' terminal. The reaction kinetics of the FQCP for Salmonella recognition is obviously improved since the probe designed with four binding sites increases the probability to react with Salmonella. As a result, the FQCP-based sensing platform can rapidly output the target detection signal within 30 min associated with a greatly improved signal-to-noise ratio up to 32.4. The system was also demonstrated with a well antimatrix effect for ultrasensitive detection of Salmonella from tap water, milk, red bull, green tea, orange juice, and Coca-Cola. Our study provides insights into the facile tailoring of functional nucleic acids for amplified and mix-to-answer detection of foodborne pathogens, which could become a powerful analytical tool for straightforward sensing of pathogens in the fields of food safety analysis, clinical diagnostics, and environmental monitoring.

Fluorescent noncanonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red- or blue-shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups, contributed either by the protein or by the substrate, that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.

Molecular dynamics plays an important role for the biological function of proteins. For protein ligand interactions, changes of conformational entropy of protein and hydration layer are relevant for the binding process. Quasielastic neutron scattering (QENS) was used to investigate differences in protein dynamics and conformational entropy of ligand-bound and ligand-free streptavidin. Protein dynamics were probed both on the fast picosecond time scale using neutron time-of-flight spectroscopy and on the slower nanosecond time scale using high-resolution neutron backscattering spectroscopy. We found the internal equilibrium motions of streptavidin and the corresponding mean square displacements (MSDs) to be greatly reduced upon biotin binding. On the basis of the observed MSDs, we calculated the difference of conformational entropy ΔSconf of the protein component between ligand-bound and ligand-free streptavidin. The rather large negative ΔSconf value (−2 kJ mol–1 K–1 on the nanosecond time scale) obtained for the streptavidin tetramer seems to be counterintuitive, given the exceptionally high affinity of streptavidin–biotin binding. Literature data on the total entropy change ΔS observed upon biotin binding to streptavidin, which includes contributions from both the protein and the hydration water, suggest partial compensation of the unfavorable ΔSconf by a large positive entropy gain of the surrounding hydration layer and water molecules that are displaced during ligand binding.

Streptavidin has applied to many areas including detection, purification, labeling, crosslinking and immobilization resulting in a high demand on its production. In this study, we report a method for preparation of recombinant core streptavidin (cSAV) protein using highperformance hydrophobic interaction chromatography (HPHIC). Firstly the cSAV was successfully cloned and expressed in Escherichia coli as inclusion bodies. A bifunctional stationary phase mainly working as HIC mode accompanied by weak anion exchange chromatography (WAX) was prepared using β-phenylethylamine (PEA) as a ligand. The denatured cSAV was then refolded and simultaneously purified by PEA hydrophobic interaction chromatography (PEA-HIC). The mass recovery and purity of cSAV by single-step were 30.2% and 98%, respectively. The bioactivity was determined to be 13.2 U/mg by biotin binding capacity assay. This method provides a new possibility for fast separation with simultaneous renaturation of cSAV.

A new strategy has been developed to provide freeze-resistant, water-soluble, and cadmium-free quantum dots made of CuInS2/ZnS, which can easily be conjugated with biomolecules. A new class of multifunctional and tetra-coordinating ligands has been synthesized based on lipoic acid, starting from lysine. The synthesized ligands not only provide facile ligand exchange of CuInS2/ZnS quantum dots but also offer an exceptional colloidal stability upon freezing, robust photophysical properties in various aqueous media across a wide pH range, and buffer composition with a tunable surface charge for the quantum dots. The quantum dots after ligand exchange is easily amenable to covalent modification with biotin binding traptavidin protein exclusively by employing simple carbodimide coupling chemistry. The quantum dots also showed excellent biocompatibility and low nonspecific binding to the cells and, thus, are suitable for live-cell imaging of cell-surface receptors.

Techniques harnessing the strong interactions of the avidin–biotin system are frequently used in biosensing and protein separation. Hydroxyapatite (Ca10(PO4)6(OH)2, HAp) is a calcium phosphate compound known to have a strong affinity for various proteins. In this study, the adsorption ability of avidin on composites and its binding activity to biotin were investigated using two types of poly(L-glutamic acid)-containing calcium phosphate composites (pGlu-HAp). The morphologies of pGlu-HAp including poly(α-glutamic acid) and poly(γ-glutamic acid) showed plate-like and sheet-like particles, respectively, due to the effects of the peptide structure. Furthermore, these particles exhibited the high adsorption capacity of avidin; the maximum adsorption amounts for poly(α-glutamic acid)-HAp and poly(γ-glutamic acid)-HAp were 776 and 302 μg mg−1 HAp, respectively. In addition, the equilibrium data for the adsorption of avidin on two pGlu-HAp-fitted Langmuir isotherm models correspond to a monolayer. The difference in the amount of adsorbed avidin on the two pGlu-HAp composites was influenced by the density of the carboxyl groups in pGlu-HAp. This revealed that avidin adsorbed on pGlu-HAp was able to bind four biotin molecules, and biotinylated protein was separated from protein mixtures by avidin-adsorbed pGlu-HAp. These results indicate that these particles would be appropriate in the development of biosensing and bioseparation tools.

We proposed and experimentally demonstrated an optofluidic biosensor using a twin-core hollow optical fiber (TC-HOF) interferometer for label-free sensing of the streptavidin-biotin binding. The TC-HOF had a central hole and two cores were imbedded near its circumference. The central hole functioned as a microfluidic channel where the liquid was flown. One of the cores was suspended at the inner surface of the hole and served as sensing arm while the other core embedded in the cladding as the reference arm, which formed an integrated all-fiber Mach-Zehnder interferometer (MZI). Both ends of TC-HOF were serially spliced to multimode fiber-single mode fiber assembly, whose lateral positions were adjusted to maximize the interference visibility. Before the surface functionalization, the pristine TC-HOF MZI showed the RI sensitivity of 2577 nm/RIU. The suspended core’s surface was chemically modified with streptavidin and biotin solutions were injected through micro-HOles in TC-HOF. Through the specific binding reaction between biotin and streptavidin, the effective refractive index (RI) around the sensing arm was modulated, which was detected by the spectral shifts in the interference patterns. The proposed sensor showed an excellent linear response to the biotin concentration in the range of 0.01–0.1 mg/mL and the net detection sensitivity of biotin was 16.9 nm/(mg/mL), which was achieved in a label-free manner. This device can be further applied to sensing and monitoring of the biochemical binding of biotin with various biomolecules such as cells, antigens, antibodies, and DNA in real-time and in situ.

Fluorescent non-canonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red or blue shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups—contributed either by the protein or substrate—that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.

Although high affinity binding between streptavidin and biotin is widely exploited, the accompanying low rate of dissociation prevents its use in many applications where rapid ligand release is also required. To combine extremely tight and reversible binding, we have introduced disulfide bonds into opposite sides of a flexible loop critical for biotin binding, creating streptavidin muteins (M88 and M112) with novel disulfide-switchable binding properties. Crystal structures reveal how each disulfide exerts opposing effects on structure and function. Whereas the disulfide in M112 disrupts the closed conformation to increase koff, the disulfide in M88 stabilizes the closed conformation, decreasing koff 260-fold relative to streptavidin. The simple and efficient reduction of this disulfide increases koff 19,000-fold, thus creating a reversible redox-dependent switch with 70-fold faster dissociation kinetics than streptavidin. The facile control of disulfide formation in M88 will enable the development of many new applications requiring high affinity and reversible binding.

Poly(dimethylsiloxane) (PDMS)-based microfluidic systems are gaining increasing attention due to their ease of fabrication, optical transparency and mechanical properties. However, the inherent hydrophobicity and chemical inertness of PDMS hinder its wider application in microfluidic systems. There is thus a strong need for methods for surface modification of PDMS-based microfluidic channels. In this work, oligo(ethylene glycol)methacrylate (OEGMA) and adamantane-containing OEGMA (OEGMA-Ada) were graft copolymerized on PDMS microchannel surfaces using a simple photochemical process to give PDMS-POA. OEGMA was chosen for its resistance to non-specific protein adsorption, and OEGMA-Ada was chosen for its subsequent attachment of mannose with bacteria binding affinity or biotin with avidin binding affinity. β-CD decorated with biotin (CD-B) and/or mannose (CD-M) was attached to the PDMS-POA microchannels via host–guest interactions between the adamantane and β-CD moieties. The data obtained suggest that the functions of the PDMS-POA/CD-B and PDMS-POA/CD-M microchannels with respect to biotin binding and bacterial adhesion were renewable. In addition, the biofunction of the PDMS-POA microchannels could be switched by treatment with SDS to release the CD component followed by treatment with a different β-CD derivative. Different from previous surface modification strategies for PDMS-based microfluidic channels, the combination of visible light-induced grafting and host–guest chemistry provides modified PDMS microchannels with renewable and switchable biofunctions for the detection and measurement of specific proteins and bacteria.

Molecular dynamics (MD) simulations were performed employing the polarized protein-specific charge (PPC) to explore the origin of the cooperativity in streptavidin–biotin systems (wild type, two single mutations and one double-mutation). The results of the experiment found that the existence of cooperativity is mainly the result of the entropic effect. In this study, the entropic contribution to the binding free energy was calculated using the recently developed interaction entropy (IE) method, and computational results are in excellent agreement with the experimental observations and are further verified by the calculation of the thermodynamic integration. Comparison of different force fields in terms of predicted binding strength ordering, cooperativity of energy and the stability of hydrogen bonding suggests that the PPC force field combined IE method is a suitable choice. In addition, the IE method enables us to obtain the residue-specific entropic contributions to the streptavidin–biotin binding affinity and identify ten hot-spot residues providing the dominant contribution to the cooperative binding. Importantly, the overall cooperativity obtained from the ten residues also comes mainly from the entropic effect in our study. The calculation of the potential of mean force shows that the unbinding of streptavidin–biotin is a multi-step process, and each step corresponds to the formation and rupture of the hydrogen bond network. And S45A mutation may increase the rigidity of the linker region, making the flap region relatively difficult to open. The present study provides significant molecular insight into the binding cooperativity of the streptavidin–biotin complex.

Monomeric streptavidin (mSA) offers a combination of structural and binding properties that are useful in many applications, including a small size and monovalent biotin binding. Because mSA contains a structurally important disulfide bond, the molecule does not fold correctly when expressed inside the cell. We show that mSA can be expressed in a functional form in Escherichia coli by fusing the OmpA signal sequence at the amino terminus. Expressed mSA is exported to the periplasm, from which the molecule leaks to the medium under vigorous shaking. Purified mSA can be conjugated with FITC and used to label microbeads and yeast cells for analysis by flow cytometry, further expanding the scope of mSA-based applications. Some applications require recombinant fusion of mSA with another protein. mSA fused to EGFP cannot be secreted to the medium but was successfully expressed in an engineered cell line that supports oxidative folding in the cytoplasm. Purified mSA-EGFP and mSA-mCherry bound biotin with high affinity and were successfully used in conventional flow cytometry and imaging flow cytometry. Finally, we demonstrate the use of mSA in ELISA, in which horseradish peroxidase-conjugated mSA and biotinylated secondary antibody are used together to detect primary antibody captured on an ELISA plate. Engineering mSA to introduce additional lysine residues can increase the reporter signal above that of wild-type streptavidin. Together, these examples establish mSA as a convenient reagent with a potentially unique role in biotechnology.

Rational design of photoreagents with systematic modifications of their structures can provide valuable information for a better understanding of the protein photocleavage mechanism by these reagents. Variation of the length of the linker connecting the photoactive moiety with the protein anchoring-group allowed us to investigate the control of the protein photocleavage site. A series of new photochemical reagents (PMA-1A, PMA-2A and PMA-3A) with increasing chain lengths is examined in the current study. Using avidin as a model system, we examined the interaction of these probes by UV–Vis, fluorescence spectroscopic methods, photocleavage and computational docking studies. Hypochromism of the absorption spectrum was observed for the binding of these new photochemical reagents with estimated binding constants (Kb) of 6.2 × 105, 6.7 × 105 and 4.6 × 105 M−1, respectively. No significant changes of Stern-Volmer quenching constant (Ksv) with Co(NH3)6Cl3 has been noted and the data indicated that the probes bind near the surface of the protein with sufficient exposure to the solvent. Photoexcitation of the probe-avidin complex, in the presence of Co(NH3)6Cl3, resulted in protein fragmentation, and the cleavage yield decreased with the increase in the linker length, and paralleled with the observed Ksv values. Amino acid sequencing of the photofragments indicated that avidin is cleaved between Thr77 and Val78, as a major cleavage site for all the three photoreagents. This site is proximate to the biotin binding site on avidin, and molecular docking studies indicated that the H-bonding interactions between the polar end-group of the photoreagents and hydrophilic amino acids of avidin were important in positioning the reagent on the protein. The major cleavage site, at residues 77–78, was within 5 A of the pyrenyl moiety of the probe, and hence, molecular tuning of the linker provided a simple approach to position the photoreagent along the potential photocleavage site.

Multivalent surface display of biomolecules is crucial to study and utilize multivalent biological interactions. However, precise valency control of surface-displayed ligands remains extremely difficult. Now a series of new oligomeric avidin proteins were fabricated that allow facile control of surface multivalency of biotinylated ligands. Naturally dimeric rhizavidin (RA) was engineered to form a mixture of oligomeric avidin assemblies, and discrete RA oligomers from the dimer to octamer of RA, were homogeneously prepared. These oligomeric avidins are in polygonal forms with expected numbers of stable biotin binding sites. Upon immobilization on low-density biotin-coated gold surfaces, RA dimer, trimer, and tetramer scaffolds provided accurate mean residual valencies of 2, 3, and 4, respectively, for biotinylated proteins. Valency-controlled display of antibody binding protein G on these RA surfaces showed clear valency-dependent enhancement of antibody capturing stability.

Small molecules regulate transcription in both eukaryotes and prokaryotes by either enhancing or repressing assembly of transcription regulatory complexes. For allosteric transcription repressors, superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, because many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. Escherichia coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria, including corepressor binding, holorepressor dimerization, and binding of the dimer to DNA. A genetic screen has yielded superrepressor mutants that repress biotin operon transcription in vivo at biotin concentrations much lower than those required by the wild type repressor. In this work, isothermal titration calorimetry and sedimentation measurements were used to determine the superrepressor biotin binding and homodimerization properties. The results indicate that, although all variants exhibit biotin binding affinities similar to that measured for BirA

To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and β domains, whereas the second consisted of a 314 amino acid from α and truncated β domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

Biotin-4-fluorescein (B4F) is a commonly used fluorescent probe for studying biotin-(strept)avidin interactions. During a characterization study of an anti-biotin antibody, using B4F as the probe, we noticed a discrepancy in the expected and experimentally determined number of biotin binding sites. Analytical testing showed that the biotin moiety in the probe undergoes a photosensitized oxidation to produce a mixture of biotin sulfoxides which has the potential to impact the quantitation of binding sites using this fluorescent probe.

Crystallization is a common practice in the purification process in small molecule synthesis while selecting the wanted product. For proteins it is rarely applied due to the methodological predicaments in obtaining crystals. Our observation of the stabilized octamers in the crystal structure of hoefavidin, a novel dimeric member of the avidin family, led to the notion of developing a novel biotechnological tool via covalent crosslinking. The avidin&ndash, biotin system has been exploited for decades utilizing the ultra-high affinity between avidin and biotin as a basis for numerous applications. Optimizing the system led to the discovery of a novel group of dimeric avidins including hoefavidin. Hoefavidin has a dynamic quaternary structure, where a dimer is the basis for generating the octamer via crystallographic symmetry operation. Upon biotin binding in solution hoefavidin dissociates solely into dimers. In order to stabilize the octamer, we designed the P61C mutant to form a disulfide bridge stabilizing the octamer and preventing dissociation upon biotin binding. The process of selecting P61C hoefavidin uniform octamers includes crystallization followed by dissolving the crystals. The P61C modified hoefavidin octamer can have a substantial added value to the various biotechnological applications and advances of the biotin based high affinity systems.

The orientation of biotin-binding sites of streptavidin adsorbed to thin films of three polythiophenes (PTs), namely, regioregular poly(3-hexylthiophene) (RP3HT), regiorandom poly(3-butylthiophene) (P3BT), and poly(3,3‴-didodecylquaterthiophene) (PQT12), has been investigated. Polymer films were examined prior to and after protein adsorption with atomic force microscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis (PCA) applied to ToF-SIMS data revealed subtle changes in surface chemistry of polymer films and orientation of adsorbed streptavidin. PCA resolved the surface alignment of alkyl side chains and differentiated the ToF-SIMS data for PQT12, RP3HT, and P3BT, verifying an amorphous morphology for P3BT and a semicrystalline one for PQT12 and RP3HT. After the characterization of the polymeric films, streptavidin adsorption from solutions with different protein concentrations (up to 300 μg/mL) has been conducted. The PCA results distinguished between amino acids characteristic for external regions of streptavidin molecules adsorbed to different PTs suggest that streptavidin adsorbed to PQT12 exposes molecular regions rich in tryptophan and tyrosine, which are components of the biotin-binding sites. The latter results were confirmed using biotin-labeled horse radish peroxidase to estimate the exposed binding sites of streptavidin adsorbed onto the different PT films. The analysis of streptavidin structure suggests that interaction between polythiophene film and dipole moment of streptavidin subunit is responsible for orientation of biotin-binding sites.

WOS: 000481724000023, This study describes the development of cryogel columns prepared by ATP having ruthenium hapten cross-linker based photosensitive approach "ANADOLUCA" for the biotinylation of Crimean Congo Hemorrhagic Fever (CCHF) primers through 5' end as a first. In this process, which does not require ATP ligand, CCHF recognition has been performed using stable and quantum dots (QDs) based avidity nanoparticles cross-linked with streptavidin. p(HEMA-co-DNA ligase) photosensitive copolymerized cryogel column as a biotinylated continuous system have prepared using ATP ligand having photosensitive ruthenium chelate based hapten cross-linker and characterized. Then, the biotinylated efficiency of the prepared column has performed through continuous biotin binding without any need of ATP ligand having to Crimean congo hemorrhagic fever primer (CCHF). The efficiency of the biotinylated CCHF primer has investigated by polymerase chain reaction (PCR). The characteristic of a sustainable biotinylating column synthesized from the same ligand that we normally develop using DNA ligase while working with ATP in the DNA ligase biotinylation process is that it can biotinize without ATP. In addition, virus-identifying ssDNA was observed as fluorescence after avidity interaction with the QD combination for viral identification after biotinylation. Then, the fluorescence intensity changes through the hybridization of 3'-ssDNA complements have determined., Bionkit Limited Company; Scientific and Technological Research Council of Turkey (TUBITAK) [1130016], This study has been partially supported by Bionkit Limited Company and Scientific and Technological Research Council of Turkey (TUBITAK) (Project number 1130016 within the scope of Tubitak 1511 Priority Areas Research Technology Development and Innovation Project Support Program).

Nanoporous anodic alumina (NAA) is a material with a high interest in nanotechnology[1]. Its physical and chemical properties combined with a cost-effective and scalable production process make it a good candidate in many nanotechnologically-related applications. Its high and tuneable surface-to-volume ratio as well as its interesting optical properties[2] have been used as the basis of different biosensing schemes[3]. Biosensing, and specifically optical biosensing is of great interest in health and environmental applications. The reflection interference spectroscopy (RIfS) method based on NAA has demonstrated its ability in detecting many kinds of molecules (proteins, antibodies[4]). One of the molecular systems with interest in biotechnological applications is the streptavidin-biotin system. The high affinity between both molecules is exploited in many applications for the binding of different biological species. For this reason, we have studied the streptavidin-biotin binding event with NAA-based RIfS, as part of a biosensing process. In our study we assess the optimal conditions for real time monitoring of the different steps (covalent streptavidin bonding, surface passivation, biotin complexation). In order to establish such optimal conditions, we have chosen the protein thrombin as a model molecule to be detected. We prepared NAA with the usual anodization conditions under oxalic acid electrolyte to obtain porous layers with uniform pore diameter and with a thickness that permits the measurement with RIfS in a flow-cell where the different species in solution can be introduced. The experiments consisted of monitoring the change in effective optical thickness (EOT, the quantitative characteristic parameter obtained from RIfS) in the different steps of the biosensing process. The NAA pore surfaces were initially functionalized with (3-aminopropyl)triethoxysilane (APTES) with the carboxyl groups activated in order to form later a peptide bond with the streptavidin. One of the parameters to be optimized was the pore diameter. Furthermore, in order to study the ability of RIfS to detect the covalent streptavidin binding to the NAA pore surface, different concentrations of streptavidin were tested. Finally, the biosensing capabilities were evaluated by detecting biotinylated thrombin at different concentrations. An example of a complete experiment with all the steps in the streptavidin-biotinylated molecule attachment detection can be seen in Figure 1. After establishing a baseline with a PBS flow, streptavidin at 50 µg/ml is introduced in the RIfS setup and its covalent attachment to APTES is detected as an increase in EOT. Following this first step, PBS is introduced to wash away the non-specifically bound molecules and a 3% Bovine Serum Albumin (BSA) solution is flowed to passivate the remaining activated carboxyl groups of APTES and the remaining hydroxyl groups on the NAA pore surface. Such steps are detected by the RIfS procedure. Then, after a subsequent PBS wash, biotinylated thrombin at a concentration of 20 µg/ml is introduced causing a steady increase in EOT reaching a value of 3.5 nm above the initial one. The experiment in Figure 1 was repeated for different NAA pore sizes revealing a higher change in EOT for bigger pore sizes. An important parameter in the detection of the biotinylated molecules binding is the concentration at the streptavidin immobilization step. Thus, different streptavidin concentrations were tested in 60 nm pore diameter NAA. Results are summarized in Figure 2: for the highest concentrations (50 µg/ml) the attachment of streptavidin shows a two-phase curve with an initial phase with a faster increase in EOT and a second phase with a slower increase after an inflection point in the EOT versus time curve. Instead, for a small concentration (1 µg/ml) an initial fast increase is followed by a stabilization of the EOT signal. These two behaviors are associated with the attachment of streptavidin to the activated carboxyl of APTES for the initial fast EOT increase, while the subsequent slower increase can be explained by the aggregation of streptavidin through amine group binding. Finally, in order to demonstrate the capability of quantifying the attachment of biotinylated molecules to the NAA surface, the process was carried out using thrombin as the model molecule and for increasing concentrations. Figure 3 shows the change in EOT as a function of the thrombin concentration. It can be seen that RIfS based on NAA sensing platforms is able to quantify such attachment. [1] Josep Ferré-Borrull, et al., Nanomaterials, vol. 7, p. 5225 (2014). [2] Josep Ferré-Borrull et al., in NANOPOROUS ALUMINA: FABRICATION, STRUCTURE, PROPERTIES AND APPLICATIONS, Springer Series in Materials Science, vol. 219, p. 185 (2015). [3] Chris Eckstein, ACS Applied Materials and Interfaces, vol. 10, p.10571 (2018). [4] Gerard Macías, Analyst, vol. 140, p. 4848 (2015). Figure 1

Graphical abstract, Highlights • Single domain antibody—rhizavidin fusion bioconjugated biotin coated quantum dots. • Provided facile and effective method to orient antibodies on QD surface. • Accomplished improved production of His-tagged rhizavidin (RZh) in E. coli. • Demonstrated utility of RZh as a replacement for tetrameric biotin binders., Straightforward and effective methods are required for the bioconjugation of proteins to surfaces and particles. Previously we demonstrated that the fusion of a single domain antibody with the biotin binding molecule rhizavidin provided a facile method to coat biotin-modified surfaces with a highly active and oriented antibody. Here, we constructed similar single domain antibody—rhizavidin fusions as well as unfused rhizavidin with a His-tag. The unfused rhizavidin produced efficiently and its utility for assay development was demonstrated in surface plasmon resonance experiments. The single domain antibody-rhizavidin fusions were utilized to coat quantum dots that had been prepared with surface biotins. Preparation of antibody coated quantum dots by this means was found to be both easy and effective. The prepared single domain antibody-quantum dot reagent was characterized by surface plasmon resonance and applied to toxin detection in a fluoroimmunoassay sensing format.

Avian influenza virus (AIV) H5N1 is a highly infectious pathogen that can be dangerous to both poultry and humans. A rapid and sensitive detection method is needed to help prevent the spread of AIV H5N1. In this study, a portable impedance biosensing system was designed to integrate a laptop computer with LabVIEW software, a data acquisition (DAQ) device, a sample delivery system, a micro-flow cell, and a gold interdigitated microelectrode (IDME) into an automatic instrument for rapid detection of AIV H5N1. Streptavidin was first immobilized onto the IDME surface, and then biotin-labeled H5N1-specific aptamer was immobilized on the IDME surface through streptavidin and biotin binding. Samples were delivered through the sample delivery system into the micro-flow cell, and AIV H5N1 was captured by the aptamer on the IDME, resulting in a change in the impedance. A virtual instrument (VI) was programmed with LabVIEW software to provide a platform for sample delivery, impedance measurement, data processing, and control. The audio card of the laptop was used as a function generator, while the DAQ device was used for data communication. The impedance measured by this biosensing system was compared with that measured by a BAS IM6 impedance analyzer, and the error was less than 5%. The results indicated that the developed system could detect AIV H5N1 at a detection limit of 0.84 HAU per 200 μL without interaction with three non-target AIV subtypes (H1N1, H5N2, and H5N3). The system was portable and automatic, and it showed great potential as a diagnostic and epidemiological tool for detection of AIV in the field.

Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells.

Techniques that are sensitive to detect mercury ion (Hg2+) are very important, due to its serious threat to public health and food security. In this work, a colorimetric aptasensor was fabricated for the detection of Hg2+ based on rolling circle amplification (RCA). The aptamer was immobilized onto the microplate and hybridized with its complementary strand (cDNA1) which linked with a primer for triggering the RCA reaction of circular template. The successfully RCA process led to the formation of long ssDNA chains on the microplate, which created many hybridized DNA fragments for bio-cDNA2. The tagged amount of horseradish peroxidase (HRP) was enhanced through the avidin/biotin binding between avi-HRP and bio-cDNA2. In the addition of TMB-H2O2, HRP was catalyzed and generated an optical signal. However, in the presence of target, Hg2+ specifically and preferentially bound with aptamer and formed a strong and stable T-Hg2+-T complex, which led to the release of cDNA1 and HRP cluster. Consequently, the optical signal decreased. Our results showed that the limit of detection (LOD) of this system was 1.6 nM with excellent specificity, and that the detection signals were enhanced by up to 18 times under RCA conditions when compared with detections without RCA. This method has been successfully used to detect Hg2+ in water samples with a recovery of 98%–105.74%.

An engineered streptavidin, SAVSBPM18 with reversible biotin binding capability, has been successfully applied to purify biotinylated and streptavidin-binding peptide (SBP) tagged proteins. To simplify the preparation for the SAVSBPM18 affinity matrix without chemical conjugation, two bio-coupling approaches were developed based on a 14-kDa dextran-binding domain (DBD) from a Leuconostoc mesenteroides dextransucrase. The first approach offers simplicity for bio-coupling by creating a direct fusion, SAVSBPM18-Linker-DBD. Purification of the fusion from crude extract and its immobilization to Sephadex can be consolidated in one-step. The second approach aims at flexibility. A SnoopCatcher (SC) was fused to DBD to create SC-Linker-DBD. This fusion can covalently capture any recombinant proteins tagged with a SnoopTag (ST) including SAVSBPM18-Linker-ST via the formation of an isopeptide bond at the interface through the SnoopCatcher-SnoopTag interaction. Although monomeric DBD binds to dextran with nanomolar affinity, DBD tetramerized via streptavidin (SAVSBPM18-Linker-ST·SC-Linker-DBD) showed an even tighter binding to Sephadex. The majority of the fluorescently labelled DBD tetramers were retained on the Sephadex surface even after four months. Affinity columns generated using either approach effectively purified both SBP-tagged and biotinylated proteins. These columns are reusable and functional even after a year of frequent use.

Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10−16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10−6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

In pretargeted radio-immunotherapy, the gradual administration of a non-radioactive tumor antigen-addressing antibody-construct and the subsequent application of a radioactive labeled, low molecular weight substance enable a highly effective and selective targeting of tumor tissue. We evaluated this concept in prostate stem cell antigen (PSCA)-positive cancers using the antigen-specific, biotinylated single chain antibody scFv(AM1)-P-BAP conjugated with tetrameric neutravidin. To visualize the systemic biodistribution, a radiolabeled biotin was injected to interact with scFv(AM1)-P-BAP/neutravidin conjugate. Biotin derivatives conjugated with different chelators for complexation of radioactive metal ions and a polyethylene glycol linker (n = 45) were successfully synthesized and evaluated in vitro and in a mouse xenograft model. In vivo, the scFv(AM1)-P-BAP showed highly PSCA-specific tumor retention with a PSCA+ tumor/PSCA- tumor accumulation ratio of ten. PEGylation of radiolabeled biotin resulted in lower liver uptake improving the tumor to background ratio.

Streptavidin, a protein produced by Streptomyces avidinii, strongly binds up to four molecules of vitamin H, d-biotin exhibiting the dissociation constant of about 10 -15 M. This strong binding affinity has been applied for detection and characterization of numerous biological molecules suggesting expression and purification of functional streptavidin should be very useful for the application of this streptavidin-biotin interaction. To express a soluble streptavidin in Escherichia coli, We synthesized streptavidin genes and cloned into pET-22b plasmid, which uses T7 RNA polymerase/T7 promoter expression systems containing pelB leader for secretion into periplasmic space and six polyhistidine tags at C-terminus for purification of expressed proteins. Although streptavidin is toxic to Escherichia coli due to strong biotin binding property, streptavidin was expressed very sufficiently in a range of 10-20 mg/ml. In SDS-PAGE, the size of purified protein was shown as 17 kDa in denatured condition (boiling) and 68 kDa in native condition (without boiling) suggesting tetramerization of monomeric subunit by non-covalent association. Further analysis by size-exclusion chromatography supported streptavidin’s tetrameric structure as well. In addition, soluble streptavidin detected biotinylated proteins in westernblot indicating its functional activity to biotin. Taken these results together, it concluded that our simple expression system was able to show high yield, homotetrameric formation and biotin binding activity analogous to natural streptavidin.

Understanding how protein oligomerization affects the stability of monomers in self-assembled structures is crucial to the development of new protein-based nanomaterials and protein cages for drug delivery. Here, we use single-molecule force spectroscopy (AFM-SMFS), protein engineering, and computer simulations to evaluate how dimerization and tetramerization affects the stability of the monomer of Streptavidin, a model homotetrameric protein. The unfolding force directly relates to the folding stability, and we find that monomer of Streptavidin is mechanically stabilized by 40% upon dimerization, and that it is stabilized an additional 24% upon tetramerization. We also find that biotin binding increases stability by another 50% as compared to the apo-tetrameric form. We used the distribution of unfolding forces to extract properties of the underlying energy landscape and found that the distance to the transition state is decreased and the barrier height is increased upon multimerization. Finally, we investigated the origin of the strengthening by ligand binding. We found that, rather than being strengthened through intramolecular contacts, it is strengthened due to the contacts provided by the biotin-binding loop that crosses the interface between the dimers.