Biotin-binding Protein Research Articles

Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin–agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.

Effects of biotinbinding proteins on eight species of pasture invertebrates

Biotinbinding proteins (BBPs) such as avidin and streptavidin represent potent insect control compounds which could be delivered via transgenic plants The effects of BBPs on some pasture pests were determined Black field cricket nymphs (Teleogryllus commodus) had significantly reduced growth and survival when fed on lettuce leaves painted with purified avidin Adult clover root weevils (Sitona lepidus) were unharmed when fed clover foliage painted with avidin In contrast neonate or oneweekold S lepidus larvae had poor survival when fed on artificial diets containing avidin or streptavidin Neonate larval Argentine stem weevils (Listronotus bonariensis) had significantly reduced survival when fed with artificial diet containing streptavidin or avidin Slugs (Deroceras reticulatum) and snails (Cantareus aspersus) were not harmed when fed with avidinpainted lettuce Similar numbers of eggs were laid and galls produced by the rootknot nematodes Meloidogyne javanica Meloidogyne hapla and Meloidogyne incognita inoculated onto transgenic tobacco plants expressing avidin and nontransgenic controls

Ligand Exchange between Proteins: EXCHANGE OF BIOTIN AND BIOTIN DERIVATIVES BETWEEN AVIDIN AND STREPTAVIDIN

We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs based on lead compounds because it is difficult to predict the structural and conformational consequences on the resultant protein-ligand interactions.

Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences.

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotin metabolism.

Effects of transgene products on honey bees (Apis mellifera) and bumblebees (Bombus sp.)

As more transgenic crop plants become commercialised, there is an increasing need for information on their impacts on honey bees and bumblebees. Direct effects on bees may arise upon ingestion of proteins encoded by transgenes, if they are expressed in pollen, nectar or resin. Indirect effects may occur if plant transformation inadvertently changes flower phenotype. This review sum- marises current findings on effects of purified transgene product ingestion on adult bee gut physiol- ogy, food consumption, olfactory learning behaviour and longevity. Bt, protease inhibitor, chitinase, glucanase and biotin-binding protein genes are discussed. Results from tests conducted in the labo- ratory with individual adult bees and with colonies in the field are presented. Observations of bee for- aging on transgenic plants kept under containment are also summarised. Results so far suggest that transgenic plant impacts on pollinators will depend on a case-by-case analysis of the gene concerned and its expression in the parts of the plant ingested by bees. Apis mellifera / Bombus terrestris / transgenic plants / Bacillus thuringiensis / protease inhibitor

Effects of Ligand−Receptor Geometry and Stoichiometry on Protein-Induced Aggregation of Biotin-Modified Colloidal Gold

Colloidal gold nanoparticles have been modified with a biotin analogue (DSDA). On addition of streptavidin, a biotin-binding protein, nanoparticle aggregation is observed. The extent and rate of aggregation depend on the relative concentrations of gold nanoparticles, DSDA, and streptavidin. To explain these effects, a model has been developed. This model is an extension of both the classical Smoluchowski model of aggregation and the recently developed model of linker induced aggregation (Kisak et al.). The inclusion of geometric and steric factors in our model explains several features that are relevant with our gold particles. The essential differences between our system and that of Kisak et al. are discussed as well as possible future areas of study.

Characterization and chromosomal localization of the chicken avidin gene family.

Chicken avidin is a biotin-binding protein expressed under inflammation in several chicken tissues and in the oviduct after progesterone induction. The gene encoding avidin belongs to a family that has been shown to include multiple genes homologous to each other. The screening and chromosomal localization studies performed to reveal the structure and organization of the complete avidin gene family is described. The avidin gene family is arranged in a single cluster within a 27-kb genomic region. The cluster is located on the sex chromosome Z on band q21. The organization of the genes was determined and two novel avidin-related genes, AVR6 and AVR7, were cloned and sequenced.

Image deconvolution for protein crystals

The image deconvolution technique developed for non-biological samples, which is based on the weak-phase-object approximation and principle of maximum entropy, was applied to simulated images of the biotin-binding protein streptavidin. A slight modification of the technique was introduced to solve the problem caused by the special image formation condition for biological samples. It has been shown that the modified technique is effective.

Tissue selective affinity targeting using the avidin-biotin system

Selective delivery of biologically active substances is designed to overcome nonspecific biodistribution of drugs and increase their local concentration at the target tissue. Certain strategies are based on carrier-mediated delivery to selective tissues and organs through ligands recognized by receptors or other address molecules on target cells. Avidins offer an attractive approach to organ- or tissue-selective targeting. Avidin and streptavidin, two biotin-binding proteins, can be targeted to specific tissues when modified with appropriate tissue markers. Their resistance to proteolytic enzymes supports long-term accumulation at the target tissue or organ, and their biotin binding sites permit the delivery of biotinylated molecules or carriers loaded with cytotoxic drugs or other bioactive substances. Modification of the two proteins with tissue-specific markers (lactose for parenchymal and trinitrophenyl for nonparenchymal liver cells) resulted in high and prolonged accumulation in the target tissue. The modified proteins could target high doses of chemotherapeutic drugs (CDDP and 5-fluorouridine) to the liver through biotinyl dextran–derived carriers. Drug Dev. Res. 50:258–271, 2000. © 2000 Wiley-Liss, Inc.

Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR.

The self-assembly of bis-biotinylated double-stranded DNA and the tetravalent biotin-binding protein streptavidin (STV) have been studied by non-denaturing gel electrophoresis and atomic force microscopy (AFM). The rapid self-assembly reproducibly generated populations of individual oligomeric complexes. Most strikingly, the oligomers predominantly contained bivalent STV molecules bridging two adjacent DNA fragments to form linear nanostructures. Trivalent STV branch points occurred with a lower frequency and the presence of tetravalent STV was scarce. However, valency distribution, size and the exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-biotinylated DNA and STV. The largest aggregates were obtained from equimolar amounts while excess STV led to the formation of smaller oligomers appearing as fingerprint-like band patterns in electrophoresis. Excess DNA, however, induces a complete breakdown of the oligomers, likely a consequence of the instability of STV conjugates containing more than two biotinylated DNA fragments. It was demonstrated that the oligomers can further be functionalized, for instance by the coupling of biotinylated immunoglobulins. Both pure and also antibody-modified DNA-STV oligomers were used as reagents in immuno-PCR (IPCR), a highly sensitive detection method for proteins and other antigens. Employment of the supramolecular reagents led to an approximately 100-fold enhanced sensitivity compared to the conventional IPCR procedure.

Affinity mode of capillary isoelectric focusing for the characterization of the biotin-binding protein actinavidin.

Different modes of capillary isoelectric focusing (CIEF) with salt mobilization and zwitterionic additive to cathodic mobilizer were applied to characterize the biotin-binding protein actinavidin and its affinity properties. The analysis is performed in a neutral coated capillary with completely eliminated electroosmotic flow. Synthetic pI standards with absorption in the visible region were used in all runs and pI determinations were based on a fitted nonlinear calibration graph. CIEF of highly purified actinavidin in native conditions was revealed in about 12 peaks in pI range 6.2-7.5, with three major forms having pI 6.80, 6.86, 6.90. CIEF of a mixture of actinavidin and increasing concentrations of two affinity ligands (biotin and biotinylated oligonucleotide) demonstrated drastic changes in the number of protein isoforms. In the latter case it resulted in only one peak (pI 5.05) when the ligand was in excess. This method, which can be named affinity CIEF, was found to be well-suited for studying structural changes, occurring in receptor proteins upon binding with a ligand. CIEF of the protein, performed under denaturing conditions (6 M urea in a solution of carrier ampholytes) is also reported. It was revealed in three isoforms with a pI more acidic than that of native actinavidin. It is demonstrated that careful selection of denaturing conditions was necessary for the reproducible results.

Coupling of antibodies with biotin.

AbstractThe avidin—biotin bond is the strongest known biological interaction between a ligand and a protein (K d=1.3×10−15 M at pH 5.0) (1). The affinity is so high that the avidin—biotm complex is extremely resistant to any type of denaturing agent (2). Biotin (Fig. 1) is a small, hydrophobic molecule that functions as a coenzyme of carboxylases (3). It is present in all living cells. Avidin is a tetrameric glycoprotein of 66,000–68,000 molecular weight, found in egg albumin and in avian tissues. The interaction between avidin and biotin occurs rapidly, and the stability of the complex has prompted its use for in situ attachment of labels in a broad variety of applications, including immunoassays, DNA hybridization (4–6), and localization of antigens in cells and tissues (7). Avidin has an isoelectric point of 10.5. Because of its positively charged residues and its oligosaccharide component, consisting mostly of mannose and glucosamine (8), avidin can interact nonspecifically with negative charges on cell surfaces and nucleic acids, or with membrane sugar receptors. At times, this causes background problems in histochemical and cytochemical applications. Streptavidin, a near-neutral, biotin-binding protein (9) isolated from the culture medium of Streptomyces avidinii, is a tetrameric nonglycosylated analog of avidin with a molecular weight of about 60,000. Like avidin, each molecule of streptavidin binds four molecules of biotin, with a similar dissociation constant. The two proteins have about 33% sequence homology, and tryptophan residues seem to be involved in their biotin binding sites (10,11). Structure of biotin. KeywordsHydrophobic MoleculeSuccinimidyl EsterSodium CyanoborohydrideBackground ProblemBiotin Binding SiteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Transmembrane insertion of the colicin Ia hydrophobic hairpin.

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an alpha-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia.

48] Competitive binding assays for biotin-binding proteins

This chapter describes the competitive binding assays for biotin-binding proteins. Radioligand binding assays of egg white avidin and egg yolk biotin-binding protein (BBP) are complicated by the presence of indeterminate amounts of biotin in samples. In each case, the exceptionally tight binding of biotin and its slow rate of dissociation mask occupied binding sites from exchange with added radiolabeled biotin. Heat-accelerated exchange to isotopic equilibrium between free and bound biotin at several sample concentrations followed by separation and measurement of protein-bound radioactivity enables the quantitation of total biotin-binding sites and the proportion of those sites occupied by endogenous biotin. A hyperbolic equation that describes the binding of radiolabeled biotin as a function of sample volume (V) at equilibrium can be transformed into the linear equation. Linearly transformed equations such as the Lineweaver–Burk equation for enzyme assays, it is convenient to apply linear regression analysis to the data to obtain values for the slopes and intercepts. However, this is statistically inappropriate because of the unequal weighting of data in double-reciprocal plots.

Protein-biotin interactions

The three-dimensional structures of several biotin-binding proteins are now known, giving insights into the molecular architecture of the binding sites for biotin. In combination with biochemical and computational approaches, these structural insights provide the basis for our present understanding of biotin—protein interactions which, in some cases, give rise to spectacular binding constants.

An oocytic membrane receptor for biotin-binding protein

The chicken oocyte accumulates a biotin-binding protein (BBP) in the yolk that is distinct from the avidin in the ‘egg white’. An identical BBP to that of yolk is also present in the circulation of the laying hen. We report the first evidence for the existence of a BBP receptor in the oocyte vitelline membrane. Reduction of the 100 kDa receptor results in loss of BBP-binding activity; this suggests that a disulfide-bonded region of the receptor is necessary for maintaining BBP-binding activity. We show further that the levels of serum BBP are strictly dependent on the presence of estrogen. As expected, BBP is not detected in significant quantities in rooster serum. Thus, these results suggest that circulatory BBP, like other estrogen-dependent components of serum, has a cognate binding activity on the oocyte membrane that may mediate its endocytosis.

Liver accumulation of TNP-modified streptavidin and avidin: potential use for targeted radio- and chemotherapy.

Hepatic metastases of malignant tumors is a major problem in the treatment of cancers for which the liver is the most common site for recurrences. In the present study we describe a selective delivery system to the liver which may facilitate specific hepatic targeting of anti-cancer agents. Avidin and streptavidin are two biotin-binding proteins with extreme resistance to proteolytic activity. Trinitrophenyl (TNP) modification of these two proteins resulted in specific accumulation in mouse liver with levels of 40-50 percent per gram tissue (%/g) during a period of several days. The two modified proteins could target to the liver high doses of covalently bound radionuclide iodine-125, a biotinylated ligand such as biotinyl-tyrosine (BT) or large biotinylated carriers such as carboxymethyl dextran (CMdex, 40kDa). Appropriately derivatized dextrans serve as carriers for various chemotherapeutic drugs, as demonstrated here for cis-dichlorodiammineplatinum (CDDP). Specific liver targeting of CDDP complexed to CMdex-TNP-streptavidin could be monitored by flame atomic absorption spectrometry of the Pt metal: High levels of the Pt drug were concentrated in the liver for at least 15hr following its targeted delivery as compared to essentially undetectable levels after administration of the free drug.

Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography

The biotin-binding protein streptavidin was crystallized as two-dimensional periodic arrays on biotinylated phospholipid monolayers. Electron diffraction patterns and images of the arrays embedded in vitreous ice were recorded to near-atomic resolution. Amplitudes and phases of structure factors were computed and combined to produce a 3 A projection density map. The reliability of the map was verified by comparing it to the available x-ray atomic model of the molecule. Projection densities from beta-strands and some amino acid side chains were identified from the electron cryomicroscopy map. These results demonstrate the first near-atomic image of this type of protein periodic array by electron crystallography, which has a great potential to aid in the structural characterization of molecular arrays engineered on a monolayer for various basic or biotechnological applications.

Analysis of the biotin-binding protein actinavidin using affinity capillary electrophoresis.

Affinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand-receptor interaction between the microbial biotin-binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio-ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio-ON, permitting the evaluation of the biotin-binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity interaction, actinavidin in the sample decreases the amount of bio-ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid-phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.