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Stefan Barth - One of the best experts on this subject based on the ideXlab platform.
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one step site specific antibody fragment auto conjugation using snap tag technology
Nature Protocols, 2019Co-Authors: Ahmad Fawzi Hussain, Florian Kampmeier, Ivo Meinholdheerlein, Paul A Heppenstall, Stefan BarthAbstract:Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site–specific conjugation methods have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme’s active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV–SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv–SNAP-tag–based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame. Fusing the SNAP-tag to a disease-specific protein of interest allows its directed functionalization with a synthetic benzylguanine-modified diagnostic or therapeutic label in a 1:1 stoichiometry. This protocol describes how to produce, conjugate and test the activity of the corresponding combination products.
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applications of snap tag technology in skin cancer therapy
Health Science Reports, 2019Co-Authors: Eden Padayachee, Henry Ademola Adeola, Jennifer C Van Wyk, Fleury Augustine Nsole Biteghe, Shivan Chetty, Nonhlanhla P Khumalo, Stefan BarthAbstract:Background Cancer treatment in the 21st century has seen immense advances in optical imaging and immunotherapy. Significant progress has been made in the bioengineering and production of immunoconjugates to achieve the goal of specifically targeting tumors. Discussion In the 21st century, antibody drug conjugates (ADCs) have been the focus of immunotherapeutic strategies in cancer. ADCs combine the unique targeting of monoclonal antibodies (mAbs) with the cancer killing ability of cytotoxic drugs. However, due to random conjugation methods of drug to antibody, ADCs are associated with poor antigen specificity and low cytotoxicity, resulting in a drug to antibody ratio (DAR) >1. This means that the cytotoxic drugs in ADCs are conjugated randomly to antibodies, by cysteine or lysine residues. This generates heterogeneous ADC populations with 0 to 8 drugs per an antibody, each with distinct pharmacokinetic, efficacy, and toxicity properties. Additionally, heterogeneity is created not only by different antibody to ligand ratios but also by different sites of conjugation. Hence, much effort has been made to find and establish antibody conjugation strategies that enable us to better control stoichiometry and site-specificity. This includes utilizing protein self-labeling tags as fusion partners to the original protein. Site-specific conjugation is a significant characteristic of these engineered proteins. SNAP-tag is one such engineered self-labeling protein tag shown to have promising potential in cancer treatment. The SNAP-tag is fused to an antibody of choice and covalently reacts specifically in a 1:1 ratio with benzylguanine (BG) substrates, eg, fluorophores or photosensitizers, to target skin cancer. This makes SNAP-tag a versatile technique in optical imaging and photoimmunotherapy of skin cancer. Conclusion SNAP-tag technology has the potential to contribute greatly to a broad range of molecular oncological applications because it combines efficacious tumor targeting, minimized local and systemic toxicity, and noninvasive assessment of diagnostic/prognostic molecular biomarkers of cancer.
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targeted delivery of dendritic polyglycerol doxorubicin conjugates by scfv snap fusion protein suppresses egfr cancer cell growth
Biomacromolecules, 2013Co-Authors: Ahmad Fawzi Hussain, Florian Kampmeier, Harald Rune Kruger, Tim Weissbach, Kai Licha, Felix Kratz, Rainer Haag, Marcelo Calderon, Stefan BarthAbstract:Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells.
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a monoclonal antibody for the detection of snap clip tagged proteins
Immunology Letters, 2013Co-Authors: Christiane Puettmann, Katharina Kolberg, Sven Hagen, Severin Schmies, Joerg Naehring, R. Fischer, Stefan BarthAbstract:SNAP/CLIP-tag technology is a novel approach that allows tagged proteins to be covalently coupled to diverse labels, such as fluorochromes and particles, using a convenient and specific enzymatic reaction. A monoclonal antibody (mAb) that binds to the SNAP/CLIP-tag would be useful to determine labeling efficiency, and to achieve reproducible detection in a variety of experimental formats. We therefore generated the murine mAb M2D11 by standard immunization and hybridoma technology. M2D11 binds to both the SNAP- and the CLIP-tag in either the coupled or uncoupled configurations and can be detected in the context of ELISA, flow cytometry, immunohistochemistry and western blot. The new antibody increases the versatility of the SNAP-tag technology by enabling the detection of tagged proteins using conventional immunological methods and widely available secondary antibodies.
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site specific covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6 o alkylguanine dna alkyltransferase
Bioconjugate Chemistry, 2009Co-Authors: Florian Kampmeier, Markus Ribbert, Thomas Nachreiner, Sofia Dembski, Arne Brecht, Florent Beaufils, Stefan BarthAbstract:Recombinant antibodies are promising tools for a wide range of bioanalytical and medical applications. However, the chemical modification of such molecules can be challenging, which limits their broader utilization. Here we describe a universal method for the site-specific labeling of antibody fragments and protein ligands by genetically fusing them to an engineered version of the human DNA-repair enzyme O(6)-alkyllguanine DNA alkyltransferase (AGT), known as SNAP-tag (1-3). Substrates containing O(6)-benzylguanine are covalently bound to the fusion proteins via a stable thioether bond in a rapid and highly specific self-labeling reaction. The coupling is site-directed, allowing the design and synthesis of antibody conjugates with predefined stoichiometry. We cloned a series of ligand SNAP-tag fusion proteins and expressed them in HEK 293T cells. The antibody/ligand-fusions were characterized by labeling with different fluorophores, labeling with biotin, or by coupling them to fluorescent nanobeads, follo...
Florian Kampmeier - One of the best experts on this subject based on the ideXlab platform.
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one step site specific antibody fragment auto conjugation using snap tag technology
Nature Protocols, 2019Co-Authors: Ahmad Fawzi Hussain, Florian Kampmeier, Ivo Meinholdheerlein, Paul A Heppenstall, Stefan BarthAbstract:Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site–specific conjugation methods have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme’s active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV–SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv–SNAP-tag–based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame. Fusing the SNAP-tag to a disease-specific protein of interest allows its directed functionalization with a synthetic benzylguanine-modified diagnostic or therapeutic label in a 1:1 stoichiometry. This protocol describes how to produce, conjugate and test the activity of the corresponding combination products.
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targeted delivery of dendritic polyglycerol doxorubicin conjugates by scfv snap fusion protein suppresses egfr cancer cell growth
Biomacromolecules, 2013Co-Authors: Ahmad Fawzi Hussain, Florian Kampmeier, Harald Rune Kruger, Tim Weissbach, Kai Licha, Felix Kratz, Rainer Haag, Marcelo Calderon, Stefan BarthAbstract:Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells.
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site specific covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6 o alkylguanine dna alkyltransferase
Bioconjugate Chemistry, 2009Co-Authors: Florian Kampmeier, Markus Ribbert, Thomas Nachreiner, Sofia Dembski, Arne Brecht, Florent Beaufils, Stefan BarthAbstract:Recombinant antibodies are promising tools for a wide range of bioanalytical and medical applications. However, the chemical modification of such molecules can be challenging, which limits their broader utilization. Here we describe a universal method for the site-specific labeling of antibody fragments and protein ligands by genetically fusing them to an engineered version of the human DNA-repair enzyme O(6)-alkyllguanine DNA alkyltransferase (AGT), known as SNAP-tag (1-3). Substrates containing O(6)-benzylguanine are covalently bound to the fusion proteins via a stable thioether bond in a rapid and highly specific self-labeling reaction. The coupling is site-directed, allowing the design and synthesis of antibody conjugates with predefined stoichiometry. We cloned a series of ligand SNAP-tag fusion proteins and expressed them in HEK 293T cells. The antibody/ligand-fusions were characterized by labeling with different fluorophores, labeling with biotin, or by coupling them to fluorescent nanobeads, follo...
Kai Johnsson - One of the best experts on this subject based on the ideXlab platform.
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A Fluorescent Sensor for GABA and Synthetic GABAB Receptor Ligands
Journal of the American Chemical Society, 2012Co-Authors: Anastasiya Masharina, Luc Reymond, Damien Maurel, Keitaro Umezawa, Kai JohnssonAbstract:While γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter, suitable tools to measure its concentration in living cells with high spatiotemporal resolution are missing. Herein, we describe the first ratiometric fluorescent sensor for GABA, dubbed GABA-Snifit, which senses GABA with high specificity and spatiotemporal resolution on the surface of living mammalian cells. GABA-Snifit is a semisynthetic fusion protein containing the GABAB receptor, SNAP- and CLIP-tag, a synthetic fluorophore and a fluorescent GABAB receptor antagonist. When assembled on cell surfaces, GABA-Snifit displays a GABA-dependent fluorescence emission spectrum in the range of 500–700 nm that permits sensing micromolar to millimolar GABA concentrations. The ratiometric change of the sensor on living cells is 1.8. Furthermore, GABA-Snifit can be utilized to quantify the relative binding affinities of GABAB receptor agonists, antagonists and the effect of allosteric modulators. These properties make GABA-Snifit a valuable ...
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a yeast based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis
Nature Chemical Biology, 2011Co-Authors: Christopher Chidley, Hirohito Haruki, Miriam Gronlund Pedersen, Evelyne Muller, Kai JohnssonAbstract:A target-identification strategy based on the yeast three-hybrid system and the SNAP-tag labeling technique identifies new targets for three small-molecule drugs and helps identify a new mechanism for the activity of the anti-inflammatory drug sulfasalazine involving inhibition of sepiapterin reductase.
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triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell a7r5
Biophysical Journal, 2010Co-Authors: Matthias Geissbuehler, Kai Johnsson, Thiemo Spielmann, Aurelie Formey, Iwan Marki, Marcel Leutenegger, Boris Hinz, Dimitri Van De VilleAbstract:The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of beta-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg(8)]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.
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An Engineered Protein Tag for Multiprotein Labeling in Living Cells
Chemistry & Biology, 2008Co-Authors: Arnaud Gautier, Maik Kindermann, Florent Beaufils, Alexandre Juillerat, Ivan R Correa, Christian Heinis, Kai JohnssonAbstract:Summary The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O 6 -benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O 2 -benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.
Ahmad Fawzi Hussain - One of the best experts on this subject based on the ideXlab platform.
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one step site specific antibody fragment auto conjugation using snap tag technology
Nature Protocols, 2019Co-Authors: Ahmad Fawzi Hussain, Florian Kampmeier, Ivo Meinholdheerlein, Paul A Heppenstall, Stefan BarthAbstract:Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site–specific conjugation methods have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme’s active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV–SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv–SNAP-tag–based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame. Fusing the SNAP-tag to a disease-specific protein of interest allows its directed functionalization with a synthetic benzylguanine-modified diagnostic or therapeutic label in a 1:1 stoichiometry. This protocol describes how to produce, conjugate and test the activity of the corresponding combination products.
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targeted delivery of dendritic polyglycerol doxorubicin conjugates by scfv snap fusion protein suppresses egfr cancer cell growth
Biomacromolecules, 2013Co-Authors: Ahmad Fawzi Hussain, Florian Kampmeier, Harald Rune Kruger, Tim Weissbach, Kai Licha, Felix Kratz, Rainer Haag, Marcelo Calderon, Stefan BarthAbstract:Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells.
Katsutoshi Hori - One of the best experts on this subject based on the ideXlab platform.
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bottom up creation of an artificial cell covered with the adhesive bacterionanofiber protein ataa
Journal of the American Chemical Society, 2019Co-Authors: Kosaku Noba, Atsuko Uyeda, Takayoshi Watanabe, Takahiro Hohsaka, Shogo Yoshimoto, Masahito Ishikawa, Tomoaki Matsuura, Katsutoshi HoriAbstract:The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In...
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bottom up creation of an artificial cell covered with the adhesive bacterionanofiber protein ataa
Journal of the American Chemical Society, 2019Co-Authors: Kosaku Noba, Atsuko Uyeda, Takayoshi Watanabe, Takahiro Hohsaka, Shogo Yoshimoto, Masahito Ishikawa, Tomoaki Matsuura, Katsutoshi HoriAbstract:The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.