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Ralf Hoffmann - One of the best experts on this subject based on the ideXlab platform.
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Additional file 12: of Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice
2019Co-Authors: Felix Fingas, Daniela Volke, Rayk Hassert, Juliane Fornefett, Sophie Funk, Christoph Baums, Ralf HoffmannAbstract:Sequence of pET21b_JF (5406âbp). Nucleotide sequence of vector pET21b_JF encoding Strep-Tag II and restrictions sites XmaI and XhoI. (PDF 116 kb
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Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Background Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. Methods The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. Results The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. Conclusions The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.
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detection of mammalian orthoreovirus type 3 reo 3 infections in mice based on serotype specific hemagglutination protein sigma 1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.
Felix Fingas - One of the best experts on this subject based on the ideXlab platform.
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Additional file 12: of Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice
2019Co-Authors: Felix Fingas, Daniela Volke, Rayk Hassert, Juliane Fornefett, Sophie Funk, Christoph Baums, Ralf HoffmannAbstract:Sequence of pET21b_JF (5406âbp). Nucleotide sequence of vector pET21b_JF encoding Strep-Tag II and restrictions sites XmaI and XhoI. (PDF 116 kb
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Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Background Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. Methods The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. Results The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. Conclusions The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.
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detection of mammalian orthoreovirus type 3 reo 3 infections in mice based on serotype specific hemagglutination protein sigma 1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.
Arne Skerra - One of the best experts on this subject based on the ideXlab platform.
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Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin
mAbs, 2014Co-Authors: Petra Kornberger, Arne SkerraAbstract:We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH 6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly 2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography--utilizing the Strep-Tag II appended to gelonin--and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC 50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.
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amination of ketones by employing two new s selective ω transaminases and the his tagged ω ta from vibrio fluvialis
European Journal of Organic Chemistry, 2012Co-Authors: Francesco G. Mutti, Arne Skerra, Christine S. Fuchs, Desiree Pressnitz, Nikolaus G. Turrini, Johann H. Sattler, Alexandra Lerchner, Wolfgang KroutilAbstract:Two recently identified (S)-selective ω-transaminases (ω-TAs) that originate from Paracoccus denitrificans (Strep-PD-ωTA, cloned with an N-terminal Strep-Tag II) and Pseudomonas fluorescens (PF-ωTA) were employed for the asymmetric amination of selected prochiral ketones. The substrates tested were transformed into optically pure amines (>99 % ee) with high conversion (up to >99 %). The ω-TAs led to higher conversion in the absence of dimethyl sulfoxide as a cosolvent than in its presence (15 %, v/v). Additionally, it was shown that a His-tagged recombinant transaminase from Vibrio fluvialis (His-VF-ωTA, cloned with an N-terminal His6-tag) showed for a single substrate, ethyl acetoacetate, significantly higher stereoselectivity for the amination compared to the corresponding commercial enzyme preparation (>99 vs. 50 %).
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fusion of a recombinant antibody fragment with a homo amino acid polymer effects on biophysical properties and prolonged plasma half life
Protein Engineering Design & Selection, 2007Co-Authors: Martin Schlapschy, Ina Theobald, Hildegard I D Mack, Margret Schottelius, Hansjurgen Wester, Arne SkerraAbstract:Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-Tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.
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The Strep-Tag system for one-step purification and high-affinity detection or capturing of proteins
Nature Protocols, 2007Co-Authors: Thomas G.m. Schmidt, Arne SkerraAbstract:The Strep-Tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-Tag II fusion proteins - including their complexes with interacting partners - both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-Tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-Tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.
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18 use of the strep tag and streptavidin for detection and purification of recombinant proteins
Methods in Enzymology, 2000Co-Authors: Arne Skerra, Thomas M SchmidtAbstract:Publisher Summary In this chapter, the Strep-Tag and streptavidin is used for the detection and purification of recombinant proteins. The Strep-Tag has the property of binding to streptavidin competitively with biotin. This behavior permits the use of very gentle conditions for the elution of a bound Strep-Tag fusion protein from the streptavidin affinity column, just by applying a diluted solution of biotin or one of its chemical derivatives. Most of the experience in the production of Strep-Tag fusion proteins has so far been gathered with E. coli as a host organism. Due to its facile and rapid genetic manipulation and its inexpensive handling, even at the fermenter scale, E. coli represents an ideal expression host, especially for highly parallelled use. This gram-negative bacterium offers two general routes for expression: (i) intracellularly in the cytosol and (ii) secretory in the periplasmic space. For both of these strategies, it must be considered that the production of a heterologous protein can be accompanied by a toxic effect on the bacterium, which may impair cell growth and plasmid stability. In respect to possible cleaning conditions for the affinity column, Strep-Tag protein purification method meets industrial standards. Because the binding of a Strep-Tag fusion protein to the affinity matrix is controlled easily and elution is effected under very mild conditions, almost every protein can be isolated in a functional state.
Petra Bielefeldt - One of the best experts on this subject based on the ideXlab platform.
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Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Background Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. Methods The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. Results The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. Conclusions The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.
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detection of mammalian orthoreovirus type 3 reo 3 infections in mice based on serotype specific hemagglutination protein sigma 1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.
Rayk Hassert - One of the best experts on this subject based on the ideXlab platform.
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Additional file 12: of Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice
2019Co-Authors: Felix Fingas, Daniela Volke, Rayk Hassert, Juliane Fornefett, Sophie Funk, Christoph Baums, Ralf HoffmannAbstract:Sequence of pET21b_JF (5406âbp). Nucleotide sequence of vector pET21b_JF encoding Strep-Tag II and restrictions sites XmaI and XhoI. (PDF 116 kb
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Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Background Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. Methods The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. Results The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. Conclusions The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.
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detection of mammalian orthoreovirus type 3 reo 3 infections in mice based on serotype specific hemagglutination protein sigma 1
Virology Journal, 2018Co-Authors: Felix Fingas, Daniela Volke, Petra Bielefeldt, Rayk Hassert, Ralf HoffmannAbstract:Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-Tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.