Investigation of Toxoplasma gondii, Neospora caninum and Tritrichomonas foetus in abortions of cattle, sheep and goats in Turkey: Analysis by real-time PCR, conventional PCR and histopathological methods.

The aim of this study was to identify Neosopora caninum, Toxoplasma gondii and Tritricomonas foetus in all cattle aborted fetus samples and N. caninum and T. gondii in sheep and goat aborted fetuses sent to Elazig Veterinary Control Institute during two years. Total genomic DNAs were obtained using a commercial kit. Real-time PCR analysis was performed separately for each agent. Conventional PCR was set up for confirmation of positive samples. Then, fetal brain, heart, lung and liver samples were analysed by hematoxylin-eosin (HE) and Avidin-Biotin Complex (ABC) Immunohistochemistry (IHC) methods. Totally, we tested 55 aborted fetus samples. Of these samples, seven (12.7 %) was belonged to goats, 18 (32.7 %) to sheep and 30 (54.5 %) to cattle. T. gondii was detected in six (10.90 %) samples, and four (7.27 %) of them were positive with Real-time PCR, while only one of these four samples was positive for both classical PCR and IHC. N. caninum was determined by at least one of the three tests in 14 (25.45 %) of the samples studied, while 8 (14.54 %) of the positive samples were detected by Real-time PCR, only two of them were also positive with conventional PCR, eight (14.54 %) samples was determined as positive by IHC. Considering T. foetus in the samples, positivity was determined in two (3.63 %) of 55 aborted fetus (both of which were aborted cattle fetus) by Real-time PCR, while only one of them was positive with conventional PCR, while no positivity was detected with the IHC.

Facile Synthesis and Characterization of a Novel Tamavidin-Luciferase Reporter Fusion Protein for Universal Signaling Applications.

Despite the avidin/biotin reaction being one of the most ubiquitous noncovalent immobilization and sensing strategies in scientific research, the ability to synthesize useful amounts of biotin-binding fusion constructs is hampered by poor solubility in bacterial expression systems. As such, there are few reports of successful genetic reporter fusions incorporating a biotin-binding partner. To address this, a sensitivity-enhanced, synthetically facile reporter fusion is developed to merge the bioluminescence output of Gaussia luciferase (Gluc) with the recently characterized biotin-binding ability of tamavidin 2 (TA2) for general and universal signaling applications in biological and analytical systems. This fusion construct enables direct bacterial expression of a reporter system incorporating two important functionalities in a 1:1 stoichiometric relationship that can provide detection of discrete events at low concentrations. Using a cold-shock expression system, highly concentrated construct can be obtained from standard culture volumes while retaining essentially native protein activity. To demonstrate feasibility and provide an example application, this fusion construct is then included in a standard target-bridged assay design for the sensitive detection of four miRNA targets.

(Strept)avidin as a template for ligands other than biotin: An overview.

Chicken avidin and bacterial streptavidin are workhorses in biotechnology. We have used avidin as a scaffold protein to develop avidin variants with novel ligand-binding affinity, so-called antidins. This article covers the strategy applied in the development of antidins. Using a phage display developed for avidin, immobilized ligands were used to select binders from a phage pool displaying avidin variants with randomized sequence in the protein loops. Antidins binding various ligands with nanomolar affinity were obtained. Antidins have already been demonstrated to be suitable for a diagnostic assay measuring serum progesterone levels and they offer a promising alternative to antibodies for the recognition of small molecules.

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

OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: 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 ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß 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). CONCLUSIONS: 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.

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

BACKGROUND: The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. RESULTS: Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. CONCLUSIONS: Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Retargeted human avidin-CAR T cells for adoptive immunotherapy of EGFRvIII expressing gliomas and their evaluation via optical imaging.

There has been significant progress in the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. However, the challenge of monitoring the therapy in real time has been continually ignored. To address this issue, we developed optical molecular imaging approaches to evaluate a recently reported novel CAR strategy for adoptive immunotherapy against glioma xenografts expressing EGFRvIII. We initially biotinylated a novel anti-EGFRvIII monoclonal antibody (biotin-4G1) to pre-target EGFRvIII+ gliomas and then redirect activated avidin-CAR expressing T cells against the pre-targeted biotin-4G1. By optical imaging study and bio-distribution analysis, we confirmed the specificity of pre-target and target and determined the optimal time for T cells adoptive transfer in vivo. The results showed this therapeutic strategy offered efficient therapy effect to EGFRvIII+ glioma-bearing mice and implied that optical imaging is a highly useful tool in aiding in the instruction of clinical CAR-T cells adoptive transfer in future.

Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects.

BACKGROUND: Wheat is considered the most important cereal crop all over the world. The wheat weevil Sitophilus granarius is a serious insect pests in much of the wheat growing area worldwide and is responsible for significant loss of yield. Avidin proteins has been proposed to function as plant defense agents against insect pests. RESULTS: A synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. The presence and expression of the transgene in six selected T0 transgenic wheat lines were confirmed at the molecular level. Accumulation of avidin protein was detected in transgenic plants compared to non-transgenic plants. Avidin transgene was stably integrated, transcribed and translated as indicated by Southern blot, ELISA, and dot blot analyses, with a high level of expression in transgenic wheat seeds. However, no expression was detected in untransformed wheat seeds. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days. CONCLUSION: Transgenic wheat plants had improved resistance to Sitophilus granarius.

Oral administration of supplementary biotin differentially influences the fertility rate and oviductal expression of avidin and avidin-related protein-2 in low- and high-fertility broiler line hens.

Probable involvement of avidin and avidin-related protein-2 (AVR2) in sperm viability in the sperm storage tubules of turkeys has been suggested. The high affinity of biotin to avidin and its analogs is also well documented. The present study aimed to determine the effect of oral biotin on reproductive performance and oviductal mRNA expression of avidin and AVR2 in 2 broiler hen lines with different fertility rates. Low-fertility (line B) and high-fertility (line D) hens (n=144) were randomly allotted to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg/L biotin in drinking water from 30 through 33 wk of age. The reproductive performance of the hens was evaluated using artificial insemination. At the end of the treatment period, 24 hens per line were killed to assay the expression of avidin and AVR2 in the uterovaginal junction. Supplementary biotin increased egg production from 73.5% for T0 to 87.8% for T2. Hens administered with biotin in line B, but not in line D, showed an increase (8.4%) in fertility rate. Hatchability, chick quality, and overall embryonic mortality were not different among the experimental groups. Real-time PCR data showed that both avidin (P=0.0013) and AVR2 (P<0.0001) expressions were influenced by a biotin×line interaction effect, where low-fertility line B hens receiving the high biotin level recorded respectively a 3.9 and 15.3% increase in avidin and AVR2 mRNA expression, although biotin did not affect these traits in line D hens. Control hens in line D had a dramatically higher AVR2 expression record (7.4-fold) compared with the control hens in line B. The correlation coefficients of fertility rate and avidin expression were 0.73 and 0.66 in lines B and D, respectively. However, the correlation of fertility and AVR2 (r=0.65) was significant for line D hens only. Overall, fertility rate and oviductal expression of avidin and AVR2 were dichotomously affected by oral biotin in low- and high-fertility line hens, where only low-fertility birds showed improvements in these attributes.

High-level expression of tamavidin 2 in human cells by codon-usage optimization.

Tamavidin 2 is a fungal protein that binds to biotin with an extremely high affinity. Tamavidin 2 is superior to avidin or streptavidin in terms of its low-level non-specific binding and high-level thermal stability. However, the gene for tamavidin 2 is highly expressed in Escherichia coli but not in mammalian cells, restricting its application as an affinity tag in mammalian cells. Here, we optimized the codon usage of tamavidin 2 for human cells and found that the resultant mutant expressed tamavidin 2 at approximately 30-fold higher level compared with the native gene. The protein thus produced in human cells could be purified by iminobiotin affinity chromatography, bound tightly to biotin, and was stable at high temperature (82 °C). This powerful technology for high-level expression of tamavidin 2 in mammalian cells will be of value in evaluating various fusion proteins produced in mammalian cells for numerous applications.

Switchavidin: reversible biotin-avidin-biotin bridges with high affinity and specificity.

Switchavidin is a chicken avidin mutant displaying reversible binding to biotin, an improved binding affinity toward conjugated biotin, and low nonspecific binding due to reduced surface charge. These properties make switchavidin an optimal tool in biosensor applications for the reversible immobilization of biotinylated proteins on biotinylated sensor surfaces. Furthermore, switchavidin opens novel possibilities for patterning, purification, and labeling.

Reproductive performance and oviductal expression of avidin and avidin-related protein-2 in young and old broiler breeder hens orally exposed to supplementary biotin.

Published data on the probable involvement of avidin and avidin-related protein-2 (AVR2) in sustaining sperm viability in sperm storage tubules in 38-wk-old turkeys, and the high affinity of avidin or its analogs to biotin suggest that supplementary biotin may increase oviductal avidin and AVR2 expression, thereby attenuating the adverse effect of aging on hen reproductive performance. Broiler breeder hens (n = 120) were randomly assigned to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg of biotin/L of drinking water from 30 to 33 (young) and 53 to 56 (old) wk of age, and artificially inseminated to determine their reproductive performance. At the end of each period of biotin administration, 8 hens from each treatment group were killed for RNA extraction from the uterovaginal junction. Egg production was lower in the old hens (44%) compared with the young ones (82%), and biotin supplementation increased egg production only in the latter. Administering supplementary biotin to young hens increased their oviductal expression of AVR2, which was much higher in the old hens (1.0 and 4.6 for young and old groups, respectively). Fertility rate was not different between young and old hens, and was increased (4.4%) at the higher level of biotin supplementation. Hatchability and hatchling quality were not affected by biotin supplementation. Embryonic mortality between 17 to 21 d of incubation was higher in young (5.2%) compared with old (1.4%) birds. Egg fertility rate showed a moderate correlation (P < 0.05) with avidin (r = 0.59) and AVR2 (r = 0.55) expression in the young-age group, and very low correlations in old-age group (0.04 and 0.17). Regardless of the hen's age, the correlation coefficient of hatchability with avidin or AVR2 expression was very low (-0.16 and 0.18). Overall, the effect of biotin supplementation on AVR2 expression, and the relationship between biotin administration and oviductal expression of avidin and AVR2 was dependent on the hen's age, being higher in the young hens.

Expression, purification, and immobilization of recombinant tamavidin 2 fusion proteins.

Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity. Unlike avidin or streptavidin, tamavidin 2 in soluble form is produced at high levels in Escherichia coli. In this chapter, we describe a method for immobilization and purification of recombinant proteins with the use of tamavidin 2 as an affinity tag. The protein fused to tamavidin 2 is tightly immobilized and simultaneously purified on biotinylated magnetic microbeads without loss of activity.

A novel chimeric avidin with increased thermal stability using DNA shuffling.

Avidins are a family of proteins widely employed in biotechnology. We have previously shown that functional chimeric mutant proteins can be created from avidin and avidin-related protein 2 using a methodology combining random mutagenesis by recombination and selection by a tailored biopanning protocol (phage display). Here, we report the crystal structure of one of the previously selected and characterized chimeric avidin forms, A/A2-1. The structure was solved at 1.8 Å resolution and revealed that the protein fold was not affected by the shuffled sequences. The structure also supports the previously observed physicochemical properties of the mutant. Furthermore, we improved the selection and screening methodology to select for chimeric avidins with slower dissociation rate from biotin than were selected earlier. This resulted in the chimeric mutant A/A2-B, which showed increased thermal stability as compared to A/A2-1 and the parental proteins. The increased stability was especially evident at conditions of extreme pH as characterized using differential scanning calorimetry. In addition, amino acid sequence and structural comparison of the chimeric mutants and the parental proteins led to the rational design of A/A2-B I109K. This mutation further decreased the dissociation rate from biotin and yielded an increase in the thermal stability.

Zebavidin--an avidin-like protein from zebrafish.

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.

Reversible biofunctionalization of surfaces with a switchable mutant of avidin.

Label-free biosensors detect binding of prey molecules (″analytes″) to immobile bait molecules on the sensing surface. Numerous methods are available for immobilization of bait molecules. A convenient option is binding of biotinylated bait molecules to streptavidin-functionalized surfaces, or to biotinylated surfaces via biotin-avidin-biotin bridges. The goal of this study was to find a rapid method for reversible immobilization of biotinylated bait molecules on biotinylated sensor chips. The task was to establish a biotin-avidin-biotin bridge which was easily cleaved when desired, yet perfectly stable under a wide range of measurement conditions. The problem was solved with the avidin mutant M96H which contains extra histidine residues at the subunit-subunit interfaces. This mutant was bound to a mixed self-assembled monolayer (SAM) containing biotin residues on 20% of the oligo(ethylene glycol)-terminated SAM components. Various biotinylated bait molecules were bound on top of the immobilized avidin mutant. The biotin-avidin-biotin bridge was stable at pH ≥3, and it was insensitive to sodium dodecyl sulfate (SDS) at neutral pH. Only the combination of citric acid (2.5%, pH 2) and SDS (0.25%) caused instantaneous cleavage of the biotin-avidin-biotin bridge. As a consequence, the biotinylated bait molecules could be immobilized and removed as often as desired, the only limit being the time span for reproducible chip function when kept in buffer (2-3 weeks at 25 °C). As expected, the high isolectric pH (pI) of the avidin mutant caused nonspecific adsorption of proteins. This problem was solved by acetylation of avidin (to pI < 5), or by optimization of SAM formation and passivation with biotin-BSA and BSA.

Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability.

A biotin-binding protein with reversible biotin-binding capability is of great technical value in the affinity purification of biotinylated biomolecules. Although several proteins, chemically or genetically modified from avidin or streptavidin, with reversible biotin-binding have been reported, they have been problematic in one way or another. Tamavidin 2 is a fungal protein similar to avidin and streptavidin in biotin-binding. Here, a mutein, tamavidin 2-REV, was engineered from tamavidin 2 by replacing the serine at position 36 (S36) with alanine. S36 is thought to form a hydrogen bond with biotin in tamavidin 2/biotin complexes and two hydrogen bonds with V38 within the protein. Tamavidin 2-REV bound to biotin-agarose and was eluted with excess free biotin at a neutral pH. In addition, the model substrate biotinylated bovine serum albumin was efficiently purified from a crude extract from Escherichia coli by means of single-step affinity chromatography with tamavidin 2-REV-immobilized resin. Tamavidin 2-REV thus demonstrated reversible biotin-binding capability. The Kd value of tamavidin 2-REV to biotin was 2.8-4.4×10(-7)M.Tamavidin 2-REV retained other convenient characteristics of tamavidin 2, such as high-level expression in E. coli, resistance to proteases, and a neutral isoelectric point, demonstrating that tamavidin 2-REV is a powerful tool for the purification of biotinylated biomolecules.

Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.

Avidin-biotin interaction mediated peptide assemblies as efficient gene delivery vectors for cancer therapy.

Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin-biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.

Targeted delivery via avidin fusion protein: intracellular fate of biotinylated doxorubicin derivative and cellular uptake kinetics and biodistribution of biotinylated liposomes.

In this study, avidin-biotin technology was combined with a multifunctional drug carrier modality i.e. liposomes to achieve an active and versatile targeting approach. The anti-cancer drug doxorubicin (DOX) was modified with direct biotinylation (B-DOX) (Allart et al., 2003), or encapsulated in biotinylated sterically stabilized pH-sensitive liposomes (BL-DOX), and targeted to the lentiviral vector transduced cells expressing an avidin fusion protein on the cell membrane (Lehtolainen et al., 2003; Lesch et al., 2009). The direct biotinylation of doxorubicin improved cell internalization in rat glioma (BT4C) cells expressing avidin fusion protein receptor but cell toxicity was reduced by 78-fold due to impaired nuclear localization. In contrast, liposomal formulations restored the biological activity of the DOX in several cell lines. However, mainly due to uptake via non-specific pathways the active targeting of BL-DOX was negligible in both in vitro and in vivo studies. Active targeting with multifunctional drug carrier systems is challenging and further studies will be needed to optimize the properties of targeted drug carrier and receptor expression systems.