Biotin-binding Activity Research Articles

Screening of biotin-binding FV-antibodies from autodisplayed FV-library on E. coli outer membrane

This study aimed to isolate FV-antibodies with biotin-binding activity from a FV-antibody library that was successfully screened on the outer membrane of E. coli. The aims were achieved by (1) preparing a library of FV-antibodies on the outer membrane of E. coli using autodisplay technology, (2) screening the FV-antibodies with biotin-binding activity from the FV-antibody library, and (3) synthesizing peptides (molecular weight of several kDa) from the biotin-binding amino acid sequence of FV-antibodies. An FV-antibody library with a diversity of 1.7 × 105 clones was prepared on the outer membrane of E. coli, using a surface display method called autodisplay technology. For the screening of biotin-binding FV-antibodies, the fluorescence-labeled biotin was introduced into the library, and the target E. coli with biotin-binding activity were screened using flow cytometry. For the screened E. coli clones, the binding affinity (KD) of Fv-antibodies against biotin was calculated and the binding properties of the screened FV-antibody were analyzed through competition assay with a synthetic peptide having the biotin-like activity. From the FRET experiment with the synthetic peptide corresponding to the CDR3 region of the screened Fv-antibody, the biotin-binding activity of the screened FV-antibody was proved to be originated from the CDR3. Finally, the applicability of the biotin-binding domain was demonstrated through the co-expression with a protein called Z-domain with antibody binding activity.

A novel cell permeability assay for macromolecules

BackgroundMany 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.ResultsWe 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.ConclusionThe assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.

Avidin-adsorbed peptide–calcium phosphate composites exhibiting high biotin-binding activity

The synthesized peptide–HAp exhibits a high adsorption capacity for avidin and a good binding ability for biotin molecules.

Recombinant streptavidin fusion proteins as signal reporters in rapid test of human hepatitis C virus infection.

Early diagnosis of hepatitis C virus (HCV) infection is very important for the treatment of the disease. Development of sensitive and specific rapid detection assays is of great significance for the diagnosis. Here, we describe a promising method of using gold-labeled streptavidin fusion proteins as novel signal reporter in a rapid detection assay for HCV infection. Recombinant genes encoding streptavidin fused with Escherichia coli maltose-binding protein (MBP) or with a portion of bacterial translational initiation factor 2 were cloned in expression vectors pMAL-5CX and pET28 and transformed in proper Escherichia coli host strains. The genes were induced and streptavidin fusion proteins, named M-STV and IF-STV, respectively, were purified by affinity chromatography to over 90% purity. The biotin-binding activity of M-STV and IF-STV was tested by enzyme-linked immunosorbent assay (ELISA). M-STV was labeled with colloidal gold nanoparticles and used as a signal reporter to develop a lateral flow-based rapid test for detecting anti-HCV antibodies in human blood samples. M-STV showed slightly higher biotin-binding activity and similar binding specificity as compared to commercial streptavidin. The gold-labeled M-STV bound specifically to biotin moieties immobilized on the rapid test strips in a dose-responsive manner and was successfully used in detecting HCV antibodies in serum samples of patients infected with HCV. The rapid test displayed higher detection sensitivity than gold-labeled commercial NeutrAvidin. Our results indicate that gold-labeled M-STV is a promising agent in rapid tests of HCV infection and possibly other viral infections.

Combinational biosynthesis and characterization of fusion proteins with tandem repeats of allophycocyanin holo-α subunits, and their application as bright fluorescent labels for immunofluorescence assay

Fusion protein of streptavidin and allophycocyanin holo-α subunit (ApcA) is fluorescent and is able to bind biotin. This fusion protein (SLA) can be used as fluorescent label in immunofluorescence assay. In this study, one or two repeats of ApcA were fused to the protein SLA, with the aim to improve its brightness. The fusion proteins SLA2 (two repeats of ApcA) and SLA3 (three repeats of ApcA), together with lyase (cpcS) and phycoerythrobilin synthesizing enzymes (Ho1 and PebS), were co-expressed in Escherichia coli. These fusion proteins were purified by affinity chromatography. While SLA2 and SLA3 shared similar absorbance spectra, fluorescence spectra and biotin-binding activities with SLA, their brightness were much higher than that of SLA. When used as fluorescent labels in immunofluorescence assay, SLA2 and SLA3 showed higher detection sensitivity than SLA. These results suggested that SLA2 and SLA3 were the preferable fluorescent labels in immunofluorescence assays.

Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

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.

A Novel Streptavidin-luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA.

A streptavidin-luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin-luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin-binding activities. It was shown that fusion has the same Km values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10-18 -10-13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin-streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin-luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.

NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching.

We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the "zero-length cross-linker" 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited ∼5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from ∼7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to ∼20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.

Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes)

A biotin-binding protein with a low isoelectric point (pI), which minimizes electrostatic non-specific binding to substances other than biotin, is potentially valuable. To obtain such a protein, we screened hundreds of mushrooms, and detected strong biotin-binding activity in the fruit bodies of Lentinula edodes, shiitake mushroom. Two cDNAs, each encoding a protein of 152 amino acids, termed lentiavidin 1 and lentiavidin 2 were cloned from L. edodes. The proteins shared sequence identities of 27%-49% with other biotin-binding proteins, and many residues that directly associate with biotin in streptavidin were conserved in lentiavidins. The pI values of lentiavidin 1 and lentiavidin 2 were 3.9 and 4.4, respectively; the former is the lowest pI of the known biotin-binding proteins. Lentiavidin 1 was expressed as a tetrameric protein with a molecular mass of 60 kDa in an insect cell-free expression system and showed biotin-binding activity. Lentiavidin 1, with its pI of 3.9, has a potential for broad applications as a novel biotin-binding protein.

Optimizing immobilization of avidin on surface-modified magnetic nanoparticles: characterization and application of protein-immobilized nanoparticles.

A simple optimization method of immobilization of avidin on magnetic nanoparticles (MNPs)' surface was proposed in this study. The avidin-immobilized MNPs were then developed and used to immobilize a model enzyme [Horseradish peroxidase (HRP)]. The loading capacity (LC) and activity of avidin-immobilized MNPs were optimized through selecting the most appropriate nanoparticle's size and shape, glutaraldehyde concentration, cross-linking reaction time, ultrasonic processing time, and initial concentration of avidin. The LC under optimized conditions was 63.37±1.29mg avidin/g MNPs, and the immobilized protein was still able to maintain its high biological activity of 10.86±0.13 U/mg (biotin-binding activity of nature avidin was 14.1U/mg) and better thermal stability compared to free avidin. A highly reusable, stable, and easily recovered immobilized HRP was obtained using MNPs as carriers. The immobilized HRP was reused repeatedly more than 9 times and retained more than 65% of its original activity.

수용성 streptavidin의 Escherichia coli 에서 기능적 발현

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.

Production and purification of streptavidin with higher biotin-binding activity

The objective of this study was to develop practical, efficient method for production, purification and assay of binding activity of streptavidin. Streptomyces avidinii was first propagated on agar plates, the bacterial cells on the agar were scrapped and suspended in a defined synthetic media (4.4 ml/cm 2 ). After 7 days agitation on a rotary shaker (200 rpm/min) at room temprature (≈28°C), the bacterial cells in the culture were pelleted. The culture supernatant was concentrated to 1/62 original volume with 75% saturation ammonium sulphate. After intensive dialysis against ammonium carbonate buffer pH 11, the suspension was loaded into an iminobiotin agarose column chromatography. The adsorbed protein (streptavidin) was eluted with sodium acetate buffer, pH 4, and the eluate was concentrated with an ultrafiltration divice and suspended in PBS. The strepatavidin-binding activity was assayed by a competitive ELISA, a competition between streptavidin in the sample and the HRP-streptavidin conjugate for the biotin (biotinyl IgG) immobilised on wells of a microtitre plate. The detection limit of this assay measured 0.16 µg/ml streptavidin. The method developed in this study produced 160 µg/ml streptavidin in the culture supernatant. After concentration with the ammonium sulphate, the streptavidin concentration increased to 4 mg/ml (69% recovery). At the final step of purification, streptavidin with 10 mg/ml concentration was obtained. The purity of the streptavidin was higher (95%) with a recovary of 19%. The purified streptavidin in this study appeared as a dimer core streotavidin on SDS PAGE and its binding activity was twice as high as that of a commercial one.

Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

Endogenous biotin or biotinylated protein binding activity is a major drawback to biotin–avidin/streptavidin detection system. The avidin/streptavidin conjugate used to detect the complex of the biotinylated secondary antibody and the primary antibody binds to endogenous biotin or biotinylated proteins leading to non-specific signals. In Western blot, the endogenous biotin or biotinylated protein binding activity is usually manifested in the form of ~72kDa, ~75kDa and ~150kDa protein bands, which often mask the signals of interest. To overcome this problem, a method based on prior hybridisation of the biotinylated secondary antibody and the streptavidin conjugate was developed. The method was tested alongside the conventional biotin–streptavidin method on proteins extracted from zebrafish (Danio rerio) embryos. Results showed that the newly developed method efficiently suppresses the endogenous biotin or biotinylated protein binding activity inherent to the biotin–streptavidin detection system.

Tamavidin 2-HOT, a highly thermostable biotin-binding protein

Tamavidin 2 is a fungal tetrameric protein that binds with high affinity to biotin, like avidin and streptavidin. We replaced asparagine-115, which lies in a subunit–subunit interface of tamavidin 2, with cysteine to generate the novel, highly thermostable protein tamavidin 2-HOT. Tamavidin 2-HOT retained more than 80% of its biotin-binding activity even after incubation at 99.9°C for 60min and was fully active in 70% dimethylsulfoxide for 30min, whereas in these harsh conditions, avidin, streptavidin, and tamavidin 2 lost their activities (less than 20% of their biotin-binding activities). The Tm in which the biotin-binding activity becomes half of tamavidin 2-HOT was 105°C, at least 20°C higher than those of avidin, streptavidin, and tamavidin 2. Because a reducing agent removed the thermal stability of tamavidin 2-HOT, the N115C mutation likely created disulfide bridges that stabilized inter-subunit associations. Tamavidin 2-HOT is efficiently produced in the soluble form by Escherichia coli for practical use. The isoelectric point of tamavidin 2-HOT (7.4) is sufficiently low to reduce the chance for non-specific binding of non-target molecules due to high positive charges. Therefore, tamavidin 2-HOT may be useful in diverse novel applications that take advantage of its high biotin-binding capability that can withstand harsh conditions.

Avidin/PSS membrane microcapsules with biotin-binding activity

Polyelectrolyte microcapsules with avidin-poly(styrene sulfonate) (PSS) membrane were prepared by a layer-by-layer deposition technique. The uptake and release of biotin-labeled fluorescein (b-FITC) as well as immobilization of biotin-labeled glucose oxidase (b-GOx) to the microcapsule were studied. The polyelectrolyte microcapsules were prepared by coating the surface of calcium carbonate (CaCO3) microparticles with an avidin/PSS multilayer membrane, followed by dissolution of CaCO3 core in an ethylenediaminetetraacetic acid solution. Inner and outer poly(allylamine)/PSS films were required to isolate the microcapsules, whereas microcapsules could not be formed without the support. The uptake of b-FITC into the microcapsule was highly enhanced through a strong binding of b-FITC to avidin as compared with the uptake of biotin-free FITC. Release of b-FITC from the microcapsule was accelerated upon addition of biotin due to a competitive binding of the added biotin to the binding site of avidin. Similarly, the surface of microcapsule was modified with b-GOx with retaining its catalytic activity.

Autodisplay of streptavidin

Streptavidin was expressed on the outer membrane of E. coli as a recombinant fusion protein with an autotransporter domain called AIDA-I (adhesin involved in diffuse adherence) using autodisplay technology. The autodisplay of streptavidin was confirmed by SDS-PAGE of the outer membrane proteins, and the number of autodisplayed streptavidin molecules on a single E. coli cell was evaluated with densitometric analysis. The biotin-binding activity of the autodisplayed streptavidin was estimated after treatment with fluorescently labeled biotin by fluorescence microscopy and flow cytometry. The biotin-binding activity of the E. coli with autodisplayed streptavidin was compared with the activity of streptavidin immobilized on magnetic beads. Finally, the outer membrane presenting autodisplayed streptavidin was isolated and layered on a 96-well microplate for an immunoassay.

On the Adsorption Behavior of Biotin-Binding Proteins on Gold and Silica

Streptavidin (SAv), avidin (Av), and neutravidin (NAv) have become widely used molecular tools in biotechnology thanks to their remarkable affinity for biotin. Their tetravalency renders these molecules particularly interesting for the functionalization of solid-liquid interfaces. Using the quartz crystal microbalance with dissipation monitoring, we systematically investigate the deposition of biotin-binding proteins to two surfaces that are popular in biotechnology: gold and silica. We find that simple physisorption of biotin-binding proteins is a viable method to confer biotin-binding functionality to gold surfaces. Both SAv and Av form dense, stable protein monolayers that retain biotin-binding activity and are largely inert to the unspecific binding of bovine serum albumin. Furthermore, we report that SAv resists adsorption to silica over a wide range of pH and ionic strength. The contrast in the binding behavior of SAv on silica and on gold suggests a simple strategy for the selective biofunctionalization of nano- or microstructured surfaces.

Four-base codon-mediated saturation mutagenesis in a cell-free translation system

Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.

Signal Peptide does not Inhibit Binding of Biotin to Streptavidin

Three recombinant polypeptides of streptavidin: the full-length streptavidin with a signal peptide (rsavS), full-length streptavidin (rsavF) and core streptavidin (rsavC), were expressed in E. coli strain BL21 (DE3) and purified by Ni-NTA chromatography. Although all three recombinant streptavidins had biotin-binding activity, the stability and solubility of rsavC tetraunits were much better than those of rsavS and rsavF, indicating that signal peptide and/or extra amino acid residues in rsavS and rsavF have negative effects on streptavidin. Meanwhile, the signal peptide and extra amino acid residues in rsavS and rsavF made it difficult for polypeptides to fold into functional proteins. After refolding of denaturing-purified proteins in vitro, both the specific activities and biotin binding sites of renatured streptavidins were 1.4-times as that of proteins obtained by native Ni-NTA purification. Because the denaturing-purified rsavC is easy of refolding into functional protein, the better strategy for production of active rsavC is to isolate the protein from IPTG-induced E. coli extracts by denaturing Ni-NTA affinity chromatography followed by refolding of purified polypeptide in vitro.

Specific delineation of BK polyomavirus in kidney tissue with a digoxigenin-labeled DNA probe

The detection of BK polyomavirus (BK virus, BKV) in kidney tissue is hampered by nonspecificity of antibodies suited to immunohistochemistry, and nonspecific background with in situ hybridization. The biotin-labeled DNA probe that is commercially available (Enzo Life Sciences, Inc.) shows good signal, but the intrinsic background in kidney tissue is high. We determined that the intrinsic background is due to endogenous biotin or biotin-binding activity in the renal tubular epithelium. Neither antibody blocking procedures nor an avidin/biotin block were entirely satisfactory for eliminating this background staining. We developed a digoxigenin-labeled DNA probe, and protocol, for detecting BK virus in formalin-fixed, paraffin embedded, kidney tissue obtained at autopsy. The hybridization signal is strong and there is no perceptible background staining. Eleven negative control kidneys all failed to hybridize. Conditions for low stringency hybridization may be employed, detecting both the related JC polyomavirus and BKV. Alternatively, high stringency hybridization conditions may be utilized, detecting BKV only. BK associated tubular necrosis is clearly demonstrated in two cases of BK nephritis.