The Experts below are selected from a list of 33165 Experts worldwide ranked by ideXlab platform
Basar Bilgicer - One of the best experts on this subject based on the ideXlab platform.
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antibody purification via affinity membrane chromatography method utilizing Nucleotide Binding Site targeting with a small molecule
Analyst, 2016Co-Authors: Nur Mustafaoglu, Tanyel Kiziltepe, Basar BilgicerAbstract:Here, we present an affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media of hybridomas and ascites fluids. The m-NBST method utilizes the Nucleotide-Binding Site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, tryptamine, which has a moderate Binding affinity to the NBS. Regenerated cellulose membrane was selected as a matrix due to multiple advantages over traditionally used resin-based affinity systems. Rituximab was used for proof of concept experiments. Antibody purification was accomplished by first capture of injected samples while running equilibration buffer (50 mM sodium phosphate pH 7.0), followed by elution achieved by running a gradient of mild elution buffer (3 M NaCl in 50 mM phosphate pH 7.0). The results indicate that the m-NBST column efficiency for Rituximab was >98%, with a purity level of >98%. The quality and the capacity of this small molecule membrane affinity purification method is further evaluated for a number of parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen Binding activity of antibodies, and column reusability and stability.
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oriented immobilization of fab fragments by Site specific biotinylation at the conserved Nucleotide Binding Site for enhanced antigen detection
Langmuir, 2015Co-Authors: Nur Mustafaoglu, Nathan J. Alves, Basar BilgicerAbstract:Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen Binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBSBiotin method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the Nucleotide Binding Site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBSBiotin method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving...
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oriented immobilization of fab fragments by Site specific biotinylation at the conserved Nucleotide Binding Site for enhanced antigen detection
Langmuir, 2015Co-Authors: Nur Mustafaoglu, Nathan J. Alves, Basar BilgicerAbstract:Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen Binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the Nucleotide Binding Site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen Binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and e-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, Site-specific biotinylation of Fab fragments without structural damage or loss in antigen Binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.
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conjugation of a reactive thiol at the Nucleotide Binding Site for Site specific antibody functionalization
Bioconjugate Chemistry, 2014Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Described here is a UV photo-cross-linking method that utilizes the NBS (Nucleotide Binding Site) for Site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBSThiol), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we Site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBSThiol method by successfully functionalizing a prostate specific antigen antibody (IgGPSA) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5–1.5 J/cm2 was determined to yield the most efficient photo-cross-linking and resulted in 1–1.5 conjugations per antibody while preserving ...
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conjugation of a reactive thiol at the Nucleotide Binding Site for Site specific antibody functionalization
Bioconjugate Chemistry, 2014Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Described here is a UV photo-cross-linking method that utilizes the NBS (Nucleotide Binding Site) for Site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we Site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen Binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of Site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of Site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.
Nathan J. Alves - One of the best experts on this subject based on the ideXlab platform.
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oriented immobilization of fab fragments by Site specific biotinylation at the conserved Nucleotide Binding Site for enhanced antigen detection
Langmuir, 2015Co-Authors: Nur Mustafaoglu, Nathan J. Alves, Basar BilgicerAbstract:Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen Binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBSBiotin method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the Nucleotide Binding Site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBSBiotin method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving...
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oriented immobilization of fab fragments by Site specific biotinylation at the conserved Nucleotide Binding Site for enhanced antigen detection
Langmuir, 2015Co-Authors: Nur Mustafaoglu, Nathan J. Alves, Basar BilgicerAbstract:Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen Binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the Nucleotide Binding Site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen Binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and e-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, Site-specific biotinylation of Fab fragments without structural damage or loss in antigen Binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.
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conjugation of a reactive thiol at the Nucleotide Binding Site for Site specific antibody functionalization
Bioconjugate Chemistry, 2014Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Described here is a UV photo-cross-linking method that utilizes the NBS (Nucleotide Binding Site) for Site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBSThiol), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we Site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBSThiol method by successfully functionalizing a prostate specific antigen antibody (IgGPSA) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5–1.5 J/cm2 was determined to yield the most efficient photo-cross-linking and resulted in 1–1.5 conjugations per antibody while preserving ...
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conjugation of a reactive thiol at the Nucleotide Binding Site for Site specific antibody functionalization
Bioconjugate Chemistry, 2014Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Described here is a UV photo-cross-linking method that utilizes the NBS (Nucleotide Binding Site) for Site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we Site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen Binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of Site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of Site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.
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oriented antibody immobilization by Site specific uv photocrosslinking of biotin at the conserved Nucleotide Binding Site for enhanced antigen detection
Biosensors and Bioelectronics, 2013Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Abstract The Nucleotide Binding Site (NBS) is an under-utilized, highly conserved Binding Site found within the variable region of nearly all antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBS Biotin ) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen Binding activity remains unaffected. An optimal UV exposure of 1 J/cm 2 yielded an average conjugation efficiency of ∼1 biotin per antibody resulting in significant immobilization efficiency while maintaining maximal antigen Binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of immobilized antibody activity to maximize assay detection capabilities. The UV-NBS Biotin method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface immobilized antibody when compared to other common immobilization methods including: e -NH 3 + surface conjugation, NHS–Biotin, and direct physical adsorption. Taken together, the UV-NBS Biotin method provides a universal, Site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies.
Tanyel Kiziltepe - One of the best experts on this subject based on the ideXlab platform.
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antibody purification via affinity membrane chromatography method utilizing Nucleotide Binding Site targeting with a small molecule
Analyst, 2016Co-Authors: Nur Mustafaoglu, Tanyel Kiziltepe, Basar BilgicerAbstract:Here, we present an affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media of hybridomas and ascites fluids. The m-NBST method utilizes the Nucleotide-Binding Site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, tryptamine, which has a moderate Binding affinity to the NBS. Regenerated cellulose membrane was selected as a matrix due to multiple advantages over traditionally used resin-based affinity systems. Rituximab was used for proof of concept experiments. Antibody purification was accomplished by first capture of injected samples while running equilibration buffer (50 mM sodium phosphate pH 7.0), followed by elution achieved by running a gradient of mild elution buffer (3 M NaCl in 50 mM phosphate pH 7.0). The results indicate that the m-NBST column efficiency for Rituximab was >98%, with a purity level of >98%. The quality and the capacity of this small molecule membrane affinity purification method is further evaluated for a number of parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen Binding activity of antibodies, and column reusability and stability.
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Selective photocrosslinking of functional ligands to antibodies via the conserved Nucleotide Binding Site.
Biomaterials, 2013Co-Authors: Nathan J. Alves, Jared F. Stefanick, Bryan F. Shaw, Michael W. Handlogten, Tanyel Kiziltepe, Matthew M. Champion, Rudolph M. Navari, Demetri T Moustakas, Basar BilgicerAbstract:Abstract The conserved Nucleotide Binding Site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized Site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for Site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS ( K d = 1–8 μ m ) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1–2 J/cm 2 yielded the most efficient photocrosslinking and resulted in 1–2 conjugations per antibody, while preserving the antigen Binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, Site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings.
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small molecule based affinity chromatography method for antibody purification via Nucleotide Binding Site targeting
Analytical Chemistry, 2012Co-Authors: Nathan J. Alves, Samuel D Stimple, Jonathan D Ashley, Michael W. Handlogten, Tanyel Kiziltepe, Basar BilgicerAbstract:The conserved Nucleotide Binding Site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized Site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a Kd ranging between 1 and 8 μM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBSIBA). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBSIBA column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBSIBA column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites flu...
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small molecule based affinity chromatography method for antibody purification via Nucleotide Binding Site targeting
Analytical Chemistry, 2012Co-Authors: Nathan J. Alves, Samuel D Stimple, Jonathan D Ashley, Michael W. Handlogten, Tanyel Kiziltepe, Basar BilgicerAbstract:The conserved Nucleotide Binding Site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized Site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a K(d) ranging between 1 and 8 μM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBS(IBA)). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBS(IBA) column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBS(IBA) column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites fluid. The results presented in this study establish the NBS(IBA) column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBS(IBA) platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost.
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oriented surface immobilization of antibodies at the conserved Nucleotide Binding Site for enhanced antigen detection
Langmuir, 2012Co-Authors: Nathan J. Alves, Tanyel Kiziltepe, Basar BilgicerAbstract:The conserved Nucleotide Binding Site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized Site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen Binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a Kd ranging from 1 to 8 μM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm2 yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen Binding activity. Comparison of the UV-NBS method with two other commonly used methods, e-NH3+ conjugation and phy...
Jianqun Chen - One of the best experts on this subject based on the ideXlab platform.
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large scale analyses of angiosperm Nucleotide Binding Site leucine rich repeat nbs lrr genes reveal three anciently diverged classes with distinct evolutionary patterns
Plant Physiology, 2016Co-Authors: Zhuqing Shao, Yueyu Hang, Yue Wu, Yanmei Zhang, Ping Wu, Bin Wang, Jianqun ChenAbstract:Nucleotide-Binding Site-leucine-rich repeat (NBS-LRR) genes make up the largest plant disease resistance gene family ( R genes), with hundreds of copies occurring in individual angiosperm genomes. However, the expansion history of NBS-LRR genes during angiosperm evolution is largely unknown. By identifying more than 6,000 NBS-LRR genes in 22 representative angiosperms and reconstructing their phylogenies, we present a potential framework of NBS-LRR gene evolution in the angiosperm. Three anciently diverged NBS-LRR classes (TNLs, CNLs, and RNLs) were distinguished with unique exon-intron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion during the first 100 million years of evolution of the angiosperm clade was observed for CNLs. TNL numbers remained stable during this period but were eventually deleted in three divergent angiosperm lineages. We inferred that an intense expansion of both TNL and CNL genes started from the Cretaceous-Paleogene boundary. Because dramatic environmental changes and an explosion in fungal diversity occurred during this period, the observed expansions of R genes probably reflect convergent adaptive responses of various angiosperm families. An ancient whole-genome duplication event that occurred in an angiosperm ancestor resulted in two RNL lineages, which were conservatively evolved and acted as scaffold proteins for defense signal transduction. Overall, the reconstructed framework of angiosperm NBS-LRR gene evolution in this study may serve as a fundamental reference for better understanding angiosperm NBS-LRR genes.
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large scale analyses of angiosperm Nucleotide Binding Site leucine rich repeat genes reveal three anciently diverged classes with distinct evolutionary patterns
Plant Physiology, 2016Co-Authors: Zhuqing Shao, Yueyu Hang, Yanmei Zhang, Bin Wang, Jiayu Xue, Jianqun ChenAbstract:Nucleotide-Binding Site-leucine-rich repeat (NBS-LRR) genes make up the largest plant disease resistance gene family (R genes), with hundreds of copies occurring in individual angiosperm genomes. However, the expansion history of NBS-LRR genes during angiosperm evolution is largely unknown. By identifying more than 6,000 NBS-LRR genes in 22 representative angiosperms and reconstructing their phylogenies, we present a potential framework of NBS-LRR gene evolution in the angiosperm. Three anciently diverged NBS-LRR classes (TNLs, CNLs, and RNLs) were distinguished with unique exon-intron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion during the first 100 million years of evolution of the angiosperm clade was observed for CNLs. TNL numbers remained stable during this period but were eventually deleted in three divergent angiosperm lineages. We inferred that an intense expansion of both TNL and CNL genes started from the Cretaceous-Paleogene boundary. Because dramatic environmental changes and an explosion in fungal diversity occurred during this period, the observed expansions of R genes probably reflect convergent adaptive responses of various angiosperm families. An ancient whole-genome duplication event that occurred in an angiosperm ancestor resulted in two RNL lineages, which were conservatively evolved and acted as scaffold proteins for defense signal transduction. Overall, the reconstructed framework of angiosperm NBS-LRR gene evolution in this study may serve as a fundamental reference for better understanding angiosperm NBS-LRR genes.
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uncovering the dynamic evolution of Nucleotide Binding Site leucine rich repeat nbs lrr genes in brassicaceae
Journal of Integrative Plant Biology, 2016Co-Authors: Zhuqing Shao, Yueyu Hang, Yanmei Zhang, Qiang Wang, Bin Wang, Jiayu Xue, Jianqun ChenAbstract:Plant genomes harbor dozens to hundreds of Nucleotide-Binding Site-leucine-rich repeat (NBS-LRR) genes; however, the long-term evolutionary history of these resistance genes has not been fully understood. This study focuses on five Brassicaceae genomes and the Carica papaya genome to explore changes in NBS-LRR genes that have taken place in this Rosid II lineage during the past 72 million years. Various numbers of NBS-LRR genes were identified from Arabidopsis lyrata (198), A. thaliana (165), Brassica rapa (204), Capsella rubella (127), Thellungiella salsuginea (88), and C. papaya (51). In each genome, the identified NBS-LRR genes were found to be unevenly distributed among chromosomes and most of them were clustered together. Phylogenetic analysis revealed that, before and after Brassicaceae speciation events, both toll/interleukin-1 receptor-NBS-LRR (TNL) genes and non-toll/interleukin-1 receptor-NBS-LRR (nTNL) genes exhibited a pattern of first expansion and then contraction, suggesting that both subclasses of NBS-LRR genes were responding to pathogen pressures synchronically. Further, by examining the gain/loss of TNL and nTNL genes at different evolutionary nodes, this study revealed that both events often occurred more drastically in TNL genes. Finally, the phylogeny of nTNL genes suggested that this NBS-LRR subclass is composed of two separate ancient gene types: RPW8-NBS-LRR and Coiled-coil-NBS-LRR.
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long term evolution of Nucleotide Binding Site leucine rich repeat genes understanding gained from and beyond the legume family
Plant Physiology, 2014Co-Authors: Zhuqing Shao, Yueyu Hang, Guangcan Zhou, Xunzong Wu, Xiaoyi Wu, Yanmei Zhang, Ping Wu, Qiang Wang, Bin Wang, Jianqun ChenAbstract:Proper utilization of plant disease resistance genes requires a good understanding of their short- and long-term evolution. Here we present a comprehensive study of the long-term evolutionary history of Nucleotide-Binding Site (NBS)-leucine-rich repeat (LRR) genes within and beyond the legume family. The small group of NBS-LRR genes with an amino-terminal RESISTANCE TO POWDERY MILDEW8 (RPW8)-like domain (referred to as RNL) was first revealed as a basal clade sister to both coiled-coil-NBS-LRR (CNL) and Toll/Interleukin1 receptor-NBS-LRR (TNL) clades. Using Arabidopsis (Arabidopsis thaliana) as an outgroup, this study explicitly recovered 31 ancestral NBS lineages (two RNL, 21 CNL, and eight TNL) that had existed in the rosid common ancestor and 119 ancestral lineages (nine RNL, 55 CNL, and 55 TNL) that had diverged in the legume common ancestor. It was shown that, during their evolution in the past 54 million years, approximately 94% (112 of 119) of the ancestral legume NBS lineages experienced deletions or significant expansions, while seven original lineages were maintained in a conservative manner. The NBS gene duplication pattern was further examined. The local tandem duplications dominated NBS gene gains in the total number of genes (more than 75%), which was not surprising. However, it was interesting from our study that ectopic duplications had created many novel NBS gene loci in individual legume genomes, which occurred at a significant frequency of 8% to 20% in different legume lineages. Finally, by surveying the legume microRNAs that can potentially regulate NBS genes, we found that the microRNA-NBS gene interaction also exhibited a gain-and-loss pattern during the legume evolution.
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a primary survey on bryophyte species reveals two novel classes of Nucleotide Binding Site nbs genes
PLOS ONE, 2012Co-Authors: Yue Wang, Letian Yang, Ping Wu, Qiang Wang, Bin Wang, Jianqun ChenAbstract:Due to their potential roles in pathogen defense, genes encoding Nucleotide-Binding Site (NBS) domain have been particularly surveyed in many angiosperm genomes. Two typical classes were found: one is the TIR-NBS-LRR (TNL) class and the other is the CC-NBS-LRR (CNL) class. It is seldom known, however, what kind of NBS-encoding genes are mainly present in other plant groups, especially the most ancient groups of land plants, that is, bryophytes. To fill this gap of knowledge, in this study, we mainly focused on two bryophyte species: the moss Physcomitrella patens and the liverwort Marchantia polymorpha, to survey their NBS-encoding genes. Surprisingly, two novel classes of NBS-encoding genes were discovered. The first novel class is identified from the P. patens genome and a typical member of this class has a protein kinase (PK) domain at the N-terminus and a LRR domain at the C-terminus, forming a complete structure of PK-NBS-LRR (PNL), reminiscent of TNL and CNL classes in angiosperms. The second class is found from the liverwort genome and a typical member of this class possesses an α/β-hydrolase domain at the N-terminus and also a LRR domain at the C-terminus (Hydrolase-NBS-LRR, HNL). Analysis on intron positions and phases also confirmed the novelty of HNL and PNL classes, as reflected by their specific intron locations or phase characteristics. Phylogenetic analysis covering all four classes of NBS-encoding genes revealed a closer relationship among the HNL, PNL and TNL classes, suggesting the CNL class having a more divergent status from the others. The presence of specific introns highlights the chimerical structures of HNL, PNL and TNL genes, and implies their possible origin via exon-shuffling during the quick lineage separation processes of early land plants.
Nur Mustafaoglu - One of the best experts on this subject based on the ideXlab platform.
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antibody purification via affinity membrane chromatography method utilizing Nucleotide Binding Site targeting with a small molecule
Analyst, 2016Co-Authors: Nur Mustafaoglu, Tanyel Kiziltepe, Basar BilgicerAbstract:Here, we present an affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media of hybridomas and ascites fluids. The m-NBST method utilizes the Nucleotide-Binding Site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, tryptamine, which has a moderate Binding affinity to the NBS. Regenerated cellulose membrane was selected as a matrix due to multiple advantages over traditionally used resin-based affinity systems. Rituximab was used for proof of concept experiments. Antibody purification was accomplished by first capture of injected samples while running equilibration buffer (50 mM sodium phosphate pH 7.0), followed by elution achieved by running a gradient of mild elution buffer (3 M NaCl in 50 mM phosphate pH 7.0). The results indicate that the m-NBST column efficiency for Rituximab was >98%, with a purity level of >98%. The quality and the capacity of this small molecule membrane affinity purification method is further evaluated for a number of parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen Binding activity of antibodies, and column reusability and stability.
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oriented immobilization of fab fragments by Site specific biotinylation at the conserved Nucleotide Binding Site for enhanced antigen detection
Langmuir, 2015Co-Authors: Nur Mustafaoglu, Nathan J. Alves, Basar BilgicerAbstract:Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen Binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBSBiotin method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the Nucleotide Binding Site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBSBiotin method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving...
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oriented immobilization of fab fragments by Site specific biotinylation at the conserved Nucleotide Binding Site for enhanced antigen detection
Langmuir, 2015Co-Authors: Nur Mustafaoglu, Nathan J. Alves, Basar BilgicerAbstract:Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen Binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the Nucleotide Binding Site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen Binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and e-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, Site-specific biotinylation of Fab fragments without structural damage or loss in antigen Binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.
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conjugation of a reactive thiol at the Nucleotide Binding Site for Site specific antibody functionalization
Bioconjugate Chemistry, 2014Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Described here is a UV photo-cross-linking method that utilizes the NBS (Nucleotide Binding Site) for Site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBSThiol), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we Site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBSThiol method by successfully functionalizing a prostate specific antigen antibody (IgGPSA) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5–1.5 J/cm2 was determined to yield the most efficient photo-cross-linking and resulted in 1–1.5 conjugations per antibody while preserving ...
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conjugation of a reactive thiol at the Nucleotide Binding Site for Site specific antibody functionalization
Bioconjugate Chemistry, 2014Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Described here is a UV photo-cross-linking method that utilizes the NBS (Nucleotide Binding Site) for Site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we Site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen Binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of Site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of Site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.
