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.     

Biochemical and Biological Characterization of a New Oxidized Avidin with Enhanced Tissue Binding Properties

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff''s base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected ¹¹¹In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.     

The 1.3 A crystal structure of a biotin-binding pseudoknot and the basis for RNA molecular recognition

A pseudoknot-containing aptamer isolated from a pool of random sequence molecules has been shown previously to represent an optimal RNA solution to the problem of binding biotin. The affinity of this RNA molecule is nonetheless orders of magnitude weaker than that of its highly evolved protein analogs, avidin and streptavidin. To understand the structural basis for biotin binding and to compare directly strategies for ligand recognition available to proteins and RNA molecules, we have determined the 1.3 A crystal structure of the aptamer complexed with its ligand. Biotin is bound at the interface between the pseudoknot's stacked helices in a pocket defined almost entirely by base-paired nucleotides. In comparison to the protein avidin, the aptamer packs more tightly around the biotin headgroup and makes fewer contacts with its fatty acid tail. Whereas biotin is deeply buried within the hydrophobic core in the avidin complex, the aptamer relies on a combination of hydrated magnesium ions and immobilized water molecules to surround its ligand. In addition to demonstrating fundamentally different approaches to molecular recognition by proteins and RNA, the structure provides general insight into the mechanisms by which RNA function is mediated by divalent metals.     

Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry.

An improved technique was developed for the unidirectional covalent binding of avidin to ferritin by reductive alkylation. The method is based on the oxidation of sugar moieties on avidin and subsequent coupling to amino groups of ferritin via Schiff's bases followed by reduction with sodium borohydride. The resultant conjugate was used as an ultrastructural marker for the localization of surface receptor sites on biotin-derivatized whole cells. Erythrocytes were treated chemically with sodium meta-periodate and biotin hydrazide in succession. The ferritin-avidin conjugates were used to label the biotin sites either before or after fixation of the cells. The density and distribution of ferritin avidin conjugates on cell surfaces were anlyzed on thin sections and compared with those of cationized ferritin, which were shown to bind anionic sites of the erythrocyte membrane. The extensions of this method for the visualization of other systems is discussed.     

Biotin binding to avidin. Oligosaccharide side chain not required for ligand association.

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.     

Syntheses of functionalized biotin N-1' derivatives: new tools for the control of gene expression with small molecules

The exceptionally high affinity of biotin toward avidin and streptavidin is at the basis of (strept)avidin-biotin biotechnology, which has numerous applications in life sciences. Recent biotin developments for in vivo and in vitro acylation of selective targeted protein and intein-mediated site specific protein biotinylation require the free biotin carboxyl function to covalently bind with the targeted protein. However, recently this carboxylic function has been used to substitute biotin with numerous ligands and flags. In the present work, we propose the N-1' labeling possibilities of biotin, keeping the valeric chain free. We describe liquid and solid-phase syntheses of functionalized biotin N-1' derivatives. Although the N-1' modification involves a two-log decrease in affinity, in vitro these molecules kept their high avidin affinity (around 10(-12) M) and the in vivo acylation ability of new biotin derivatives.     

Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs

DNA labeled with the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP was chromatographed on biotin cellulose affinity columns using either avidin or streptavidin as the affinity reagent. Although both proteins were equally effective in binding the Bio-12-SS-DNA to the affinity resin, two important differences were found. First, nonbiotinylated DNA bound to avidin, but not to streptavidin, in buffers containing 50 mM NaCl. Second, Bio-12-SS-DNA was released much more slowly from the streptavidin affinity column than from the avidin column upon washing with buffer containing dithiothreitol. This difficulty in reducing the disulfide bond of Bio-12-SS-DNA bound to streptavidin is most likely due to steric protection of the disulfide bond by the protein. This conclusion is supported by our finding that DNA labeled with another biotinylated nucleotide analog, Bio-19-SS-dUTP, is rapidly and efficiently recovered from a streptavidin column. In Bio-19-SS-DNA, the distance between the disulfide bond and the biotin group is approximately 10 A greater than that in Bio-12-SS-DNA. Therefore, Bio-19-SS-dUTP and streptavidin form the basis of an efficient affinity system for the isolation and subsequent recovery of biotinylated DNA in the presence of low ionic strength buffers.     

Avidin-like domain in an epidermal growth factor homolog from a sea urchin

We have found that a protein from the purple sea urchin has a carboxyl-terminal domain with striking sequence similarity to chicken avidin and bacterial streptavidin. All our evidence supports the homology of these sequences. Tetramers of avidin and streptavidin bind biotin strongly; the biotin binding site involves two to four tryptophans and probably an adjacent lysine in each chain. The presence of four tryptophans at equivalent positions in the sea urchin protein domain suggests that it may also be able to bind biotin and inhibit cell growth, as do the two other proteins. Alternatively, this domain may have acquired a new role as part of a multidomain protein.     

Recognition of chemically damaged DNA by the gene 32 protein from bacteriophage T4

We have used fluorescence spectroscopy to investigate the binding of gene 32 protein from bacteriophage T4 to DNA which has been chemically modified with carcinogens or antitumor drugs. This protein exhibits a high specificity for single-stranded nucleic acids and binds more efficiently to DNA modified either with cis-diaminodichloroplatinum(II) or with aminofluorene derivatives than to native DNA. This increased affinity is related to the formation of locally unpaired regions which are strong binding sites for the single-strand binding protein. In contrast, gene 32 protein has the same affinity for native DNA, DNA containing methylated purines and DNA that has reacted with trans-diaminodichloroplatinum(II) or with chlorodiethylenetriaminoplatinum(II) chloride. These types of damage do not induce a sufficient structural change to allow gene 32 protein binding. Depurination of DNA does not create binding sites for the T4 gene 32 protein but nicked apurinic sites are strong ligands for the protein. This T4 single-strand binding protein does not exhibit a significantly increased affinity for nicked DNA as compared with native DNA. These results are discussed with respect to the recognition of DNA damage by proteins involved in DNA repair and to the possible role of single-strand binding proteins in DNA repair mechanisms.     

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.     

Label-free amplified bioaffinity detection using terahertz wave technology

A new affinity biosensor based on pulsed terahertz (THz) wave technology has been used to monitor binding between biotin and avidin molecules. Amplified detection of avidin-biotin binding is obtained on supported membranes composed of biotin layers on quartz surface, which is modified with octadecanol. Agarose particles are conjugated with avidin and then applied to biotin, which is already bound to the octadecanol quartz surface, the biotin binds to the conjugate rapidly and causes an enhancement of the THz difference signal between biotin and biotin-avidin complexes by a factor greater than eight fold when compared to the same sample without agarose beads. The technique was able to detect less than 10.3 ng/cm2 avidin, thus, giving the THz system a detection capability of sub-thin solid films better than ellipsometry and reflectometry techniques. Further improvement is underway using highly refractive beads together with appropriate surface chemistry. This newly developed method is being saliently optimized for future application, including the detection of DNA hybridization and ligand-analyte affinity binding.     

Probing the adenosine receptor with adenosine and xanthine biotin conjugates

Biotin-containing analogs of a potent agonist (N6-phenyladenosine) and a potent antagonist (1,3-dipropyl-8-phenylxanthine) of adenosine receptor activity have been synthesized. A spacer chain to the biotin moiety is attached in both cases to the para-position of the phenyl ring. Two biotin conjugates of N6-phenyladenosine differing only in the length of the spacer chain bind to the adenosine receptor and to avidin simultaneously. The shorter-chain derivative was more potent in inhibiting binding of N6-[3H]cyclohexyladenosine to rat cerebral cortical membranes (Ki of 11 nM in the absence of avidin, 36 nM for the avidin complex). Three biotin conjugates of 1,3-dipropyl-8-phenylxanthine bound competitively to the adenosine receptor, but only in the absence of avidin. The results are interpreted in terms of the possible orientation of the ligands at the receptor binding site.     

Dual-affinity avidin molecules

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.     

Optimisation of culture conditions with respect to biotin requirement for the production of recombinant avidin in Pichia pastoris

Due to its very high affinity to biotin, avidin is one of the most widely exploited proteins in modern biotechnological and biomedical applications. Since biotin is an essential vitamin for the growth of many microorganisms, we examined the effect of biotin deficiency on growth for a recombinant Pichia pastoris strain expressing and secreting a recombinant glycosylated avidin. The results showed that biotin deficiency lowers growth rate and biomass yield for P. pastoris. Substitution of biotin in the medium by the two structurally unrelated compounds, aspartic acid and oleic acid, which do not bind to recombinant avidin was analyzed quantitatively. These two compounds had a growth promoting effect in biotin-deficient medium, but did not replace biotin completely. Indeed, in chemostat culture, wash-out occurred after about six liquid residence times and recombinant avidin productivity was lowered. However, addition of low amounts of biotin (20 microg L(-1) of biotin for a cell density of 8 g L(-1)) resulted in stable chemostat cultures on methanol with the production of recombinant biotin-free avidin. The specific avidin production rate was 22 microg g(-1) h(-1) at a dilution rate of 0.06 h(-1).     

A modular design of low‐background bioassays based on a high‐affinity molecular pair barstar:barnase

High‐affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K_D) of the molecular pair, with avidin:biotin being the state‐of‐the‐art molecular pair (K_D ～ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high‐affinity protein pair, barstar:barnase (K_D ～ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non‐negligible background due to the non‐specific binding. A quantitative assessment of the non?specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin‐based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non‐specific binding, showing good prospects for high‐sensitivity rare biomolecular event nanoproteomic assays.     

Evidence for the biosynthesis of selenobiotin

Chemically synthesized selenobiotin is, like sulfur biotin, able to bind to avidin. This observation was used to help identify biologically synthesized selenobiotin as an excretion product of $\text{Phycomyces blakesleeanus}$. The identification of [⁷⁵Se]selenobiotin was based on the highly specific binding of biotin to avidin used as an affinity ligand to Sepharose, on its release from the complex by proteolytic treatment, and its chromatographic behavior relative to [¹⁴C]biotin standards. These results represent the first evidence of a biological synthesis of a heterocyclic ring that contains selenium in place of sulfur.     

Bifunctional avidin with covalently modifiable ligand binding site

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

Binding specificity of the two major DNA-binding proteins in human serum.

The two major DNA-binding proteins of human serum (DNA-binding protein 1 and DNA-binding protein 2) were shown to bind preferentially to single-stranded polynucleotides rich in guanine residues. Equilibrium competition experiments using a nitrocellulose filter assay system containing labeled human lymphocyte DNA and various competing natural and synthetic polynucleotides indicated that both proteins recognized sequences of bases containing a keto group in either position 6 (purines) or 4 (pyrimidines) and that these keto groups must be readily accessible for effective binding to occur. Guanine was shown to be the preferred nucleotide through inhibition experiments using a series of synthetic homopolymers and a series of bacterial DNAs of differing G + C content. The relationship between protein affinity and G + C content was shown to be directly proportional. The equilibrium constants for the binding of the human lymphocyte DNA by both proteins were on the order of 10(-6) M, and the length of the nucleotide sequence necessary for effective binding was found to be 12 to 18 bases using a series of oligomers of poly(dG).     

抗生物素蛋白结合蛋白的发现及分离纯化

本文报道了在日本血吸虫感染的兔血清及日本血吸虫卵中,存在一种能与抗生物素蛋白（又称亲和素）专一性结合的蛋白质,称为抗生物素蛋白结合蛋白。该蛋白质与亲和素的结合与生物素及亲和素的糖链部分无关,并能在高ＰＨ、高浓度盐及去污剂等条件下与亲和素结合。利用ＤＥＡ－５２离子交换柱及偶联有亲和素的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱,从日本血吸虫感染的兔血清中,分离纯化了该蛋白质。提纯的亲和素结合蛋白在ＳＤＳ－ＰＡ     

Role of structure and dynamics of DNA with cisplatin and oxaliplatin adducts in various sequence contexts on binding of HMGB1a

Anti-cancer drugs, such as cisplatin and oxaliplatin, covalently bind to adjacent guanine bases in DNA to form intra-strand adducts. Differential recognition of drug–DNA adducts by the protein HMGB1a has been related to the differences in efficacy of these drugs in tumours. Additionally, the bases flanking the adduct (the sequence context) also have a marked effect on HMGB1a binding affinity. We perform atomistic molecular dynamics simulations of DNA with cisplatin and oxaliplatin adducts in four sequence contexts (AGGC, CGGA, TGGA and TGGT) in the absence and presence of HMGB1a. The structure of HMGB1a-bound drug–DNA molecules is independent of sequence and drug identity, confirming that differential recognition cannot be explained by the protein-bound structure. The differences in the static and conformational dynamics of the drug–DNA structures in the absence of the protein explain some but not all trends in differential binding affinity of HMGB1a. Since the minor groove width and helical bend of all drug–DNA molecules in the unbound state are lower than the protein-bound state, HMGB1a must actively deform the DNA during binding. The thermodynamic pathway between the unbound and protein-bound states could be an additional factor in the binding affinity of HMGB1a for drug–DNA adducts in various sequence contexts.