The Experts below are selected from a list of 22914 Experts worldwide ranked by ideXlab platform
Michael Hust - One of the best experts on this subject based on the ideXlab platform.
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Discovery of Leptospira spp. seroreactive peptides using ORFeome Phage Display
PLoS Neglected Tropical Diseases, 2019Co-Authors: Siti Roszilawati Ramli, Jonas Zantow, Van Kinh Nguyen, Natalia Novoselova, Gustavo Marçal Schmidt Garcia Moreira, Frank Pessler, Marga G A Goris, Michael HustAbstract:Background Leptospirosis is the most common zoonotic disease worldwide. The diagnostic performance of a serological test for human leptospirosis is mainly influenced by the antigen used in the test assay. An ideal serological test should cover all serovars of pathogenic leptospires with high sensitivity and specificity and use reagents that are relatively inexpensive to produce and can be used in tropical climates. Peptide-based tests fulfil at least the latter two requirements, and ORFeome Phage Display has been successfully used to identify immunogenic peptides from other pathogens. Methodology/Principal findings Two ORFeome Phage Display libraries of the entire Leptospira spp. genomes from five local strains isolated in Malaysia and seven WHO reference strains were constructed. Subsequently, 18 unique Leptospira peptides were identified in a screen using a pool of sera from patients with acute leptospirosis. Five of these were validated by titration ELISA using different pools of patient or control sera. The diagnostic performance of these five peptides was then assessed against 16 individual sera from patients with acute leptospirosis and 16 healthy donors and was compared to that of two recombinant reference proteins from L. interrogans. This analysis revealed two peptides (SIR16-D1 and SIR16-H1) from the local isolates with good accuracy for the detection of acute leptospirosis (area under the ROC curve: 0.86 and 0.78, respectively; sensitivity: 0.88 and 0.94; specificity: 0.81 and 0.69), which was close to that of the reference proteins LipL32 and Loa22 (area under the ROC curve: 0.91 and 0.80; sensitivity: 0.94 and 0.81; specificity: 0.75 and 0.75). Conclusions/Significance This analysis lends further support for using ORFeome Phage Display to identify pathogen-associated immunogenic peptides, and it suggests that this technique holds promise for the development of peptide-based diagnostics for leptospirosis and, possibly, of vaccines against this pathogen.
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ORFeome Phage Display.
Methods of Molecular Biology, 2017Co-Authors: Jonas Zantow, Gustavo Marçal Schmidt Garcia Moreira, Stefan Dübel, Michael HustAbstract:ORFeome Phage Display allows the efficient functional screening of entire proteomes or even metaproteomes to identify immunogenic proteins. For this purpose, randomly fragmented, whole genomes or metagenomes are cloned into a Phage-Display vector allowing positive selection for open reading frames (ORF) to improve the library quality. These libraries Display all possible proteins encoded by a pathogen or a microbiome on the Phage surface. Consequently, immunogenic proteins can be selected from these libraries using disease-related immunoglobulins from patient serum. ORFeome Phage Display in particular allows the identification of immunogenic proteins that are only expressed in the host-pathogen interaction but not in cultivation, as well as the detection of very low expressed and very small immunogens and immunogenic proteins of non-cultivable organisms. The identified immunogenic proteins are potential biomarkers for the development of diagnostic assays or vaccines. These articles will give an introduction to ORFeome Phage-Display technology and give detailed protocols to identify immunogenic proteins by Phage Display.
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Designing Human Antibodies by Phage Display
Transfusion Medicine and Hemotherapy, 2017Co-Authors: Anne C. Frenzel, Doris Meier, Saskia Helmsing, Thomas Schirrmann, Jonas Kügler, André Frenzel, Michael Hust, Stefan DübelAbstract:With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by Phage Display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.
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Phage Display derived human antibodies in clinical development and therapy
mAbs, 2016Co-Authors: André Frenzel, Thomas Schirrmann, Michael HustAbstract:ABSTRACTOver the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity.Antibody Phage Display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by Phage Display were approved for therapy. In 2002, ada...
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Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by Phage Display.
Proteomics Clinical Applications, 2016Co-Authors: Philipp Kuhn, Sebastian Miethe, Gustavo Marçal Schmidt Garcia Moreira, Viola Fühner, Tobias Unkauf, André Frenzel, Michael HustAbstract:Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody Phage Display to generate recombinant antibodies. This in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of antibodies against all conceivable molecules. Phage Display technology enables obtaining human antibodies from naive antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune Phage Display libraries to select in vivo affinity-matured antibodies. Because the Phage packaged DNA sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of Phage Display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.
Andreas Plückthun - One of the best experts on this subject based on the ideXlab platform.
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efficient selection of darpins with sub nanomolar affinities using srp Phage Display
Journal of Molecular Biology, 2008Co-Authors: Daniel Steiner, Patrik Forrer, Andreas PlückthunAbstract:There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP Phage Display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous Phage Display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional Phage Display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome Display. Here, we harnessed SRP Phage Display to generate a Phage DARPin library containing more than 10(10) individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFalpha, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP Phage Display makes filamentous Phage Display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP Phage Display will be beneficial for other libraries of stable and fast-folding proteins.
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signal sequences directing cotranslational translocation expand the range of proteins amenable to Phage Display
Nature Biotechnology, 2006Co-Authors: Daniel Steiner, Patrik Forrer, Michael T Stumpp, Andreas PlückthunAbstract:Signal sequences directing cotranslational translocation expand the range of proteins amenable to Phage Display
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Signal sequences directing cotranslational translocation expand the range of proteins amenable to Phage Display
Nature Biotechnology, 2006Co-Authors: Daniel Steiner, Patrik Forrer, Michael T Stumpp, Andreas PlückthunAbstract:Even proteins that fold well in bacteria are frequently Displayed poorly on filamentous Phages. Low protein presentation on Phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in Phage assembly, we tested the Display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher Display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the Display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved Phage Display from tenfold enrichment to >1,000-fold enrichment per Phage Display selection round. We named this method 'SRP Phage Display' and envision broad applicability, especially when Displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.
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selection for improved protein stability by Phage Display
Journal of Molecular Biology, 1999Co-Authors: Sabine Jung, Annemarie Honegger, Andreas PlückthunAbstract:A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, Phage Display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the Display Phages at elevated temperatures was compared to exposure of the Phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in Phage Display. # 1999 Academic Press
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Selection for improved protein stability by Phage Display
Journal of Molecular Biology, 1999Co-Authors: Sabine Jung, Annemarie Honegger, Andreas PlückthunAbstract:A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, Phage Display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the Display Phages at elevated temperatures was compared to exposure of the Phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in Phage Display.
Thomas Schirrmann - One of the best experts on this subject based on the ideXlab platform.
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Designing Human Antibodies by Phage Display
Transfusion Medicine and Hemotherapy, 2017Co-Authors: Anne C. Frenzel, Doris Meier, Saskia Helmsing, Thomas Schirrmann, Jonas Kügler, André Frenzel, Michael Hust, Stefan DübelAbstract:With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by Phage Display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.
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Phage Display derived human antibodies in clinical development and therapy
mAbs, 2016Co-Authors: André Frenzel, Thomas Schirrmann, Michael HustAbstract:ABSTRACTOver the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity.Antibody Phage Display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by Phage Display were approved for therapy. In 2002, ada...
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Phage Display-derived human antibodies in clinical development and therapy
mAbs, 2016Co-Authors: Anne C. Frenzel, Thomas Schirrmann, André Frenzel, Michael HustAbstract:Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody Phage Display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by Phage Display were approved for therapy. In 2002, adalimumab (Humira?)becamethe first Phage Display-derived antibody granted a marketing approval. Humira? was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous Phage Display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, Phage Display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about Phage Display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the Phage Display technology and its development over the last 25 years.
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generation and analysis of the improved human hal9 10 antibody Phage Display libraries
BMC Biotechnology, 2015Co-Authors: Jonas Kügler, Doris Meier, Henk Garritsen, Florian Tomszak, Sonja Wilke, Thomas Schirrmann, Stefan Dübel, André Frenzel, Bjorn Hock, Lars ToleikisAbstract:Antibody Phage Display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody Phage Display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved Phage Display vectors. In detail, most common Phagemids employed for antibody Phage Display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody Phage Display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new Phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or Phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv Phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.
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generation and analysis of the improved human hal9 10 antibody Phage Display libraries
BMC Biotechnology, 2015Co-Authors: Jonas Kügler, Doris Meier, Henk Garritsen, Florian Tomszak, Sonja Wilke, Thomas Schirrmann, Stefan Dübel, André Frenzel, Bjorn Hock, Lars ToleikisAbstract:Background Antibody Phage Display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody Phage Display library.
André Frenzel - One of the best experts on this subject based on the ideXlab platform.
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Designing Human Antibodies by Phage Display
Transfusion Medicine and Hemotherapy, 2017Co-Authors: Anne C. Frenzel, Doris Meier, Saskia Helmsing, Thomas Schirrmann, Jonas Kügler, André Frenzel, Michael Hust, Stefan DübelAbstract:With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by Phage Display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.
-
Phage Display derived human antibodies in clinical development and therapy
mAbs, 2016Co-Authors: André Frenzel, Thomas Schirrmann, Michael HustAbstract:ABSTRACTOver the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity.Antibody Phage Display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by Phage Display were approved for therapy. In 2002, ada...
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Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by Phage Display.
Proteomics Clinical Applications, 2016Co-Authors: Philipp Kuhn, Sebastian Miethe, Gustavo Marçal Schmidt Garcia Moreira, Viola Fühner, Tobias Unkauf, André Frenzel, Michael HustAbstract:Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody Phage Display to generate recombinant antibodies. This in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of antibodies against all conceivable molecules. Phage Display technology enables obtaining human antibodies from naive antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune Phage Display libraries to select in vivo affinity-matured antibodies. Because the Phage packaged DNA sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of Phage Display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.
-
Phage Display-derived human antibodies in clinical development and therapy
mAbs, 2016Co-Authors: Anne C. Frenzel, Thomas Schirrmann, André Frenzel, Michael HustAbstract:Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody Phage Display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by Phage Display were approved for therapy. In 2002, adalimumab (Humira?)becamethe first Phage Display-derived antibody granted a marketing approval. Humira? was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous Phage Display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, Phage Display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about Phage Display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the Phage Display technology and its development over the last 25 years.
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generation and analysis of the improved human hal9 10 antibody Phage Display libraries
BMC Biotechnology, 2015Co-Authors: Jonas Kügler, Doris Meier, Henk Garritsen, Florian Tomszak, Sonja Wilke, Thomas Schirrmann, Stefan Dübel, André Frenzel, Bjorn Hock, Lars ToleikisAbstract:Antibody Phage Display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody Phage Display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved Phage Display vectors. In detail, most common Phagemids employed for antibody Phage Display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody Phage Display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new Phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or Phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv Phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.
Daniel Steiner - One of the best experts on this subject based on the ideXlab platform.
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efficient selection of darpins with sub nanomolar affinities using srp Phage Display
Journal of Molecular Biology, 2008Co-Authors: Daniel Steiner, Patrik Forrer, Andreas PlückthunAbstract:There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP Phage Display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous Phage Display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional Phage Display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome Display. Here, we harnessed SRP Phage Display to generate a Phage DARPin library containing more than 10(10) individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFalpha, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP Phage Display makes filamentous Phage Display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP Phage Display will be beneficial for other libraries of stable and fast-folding proteins.
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signal sequences directing cotranslational translocation expand the range of proteins amenable to Phage Display
Nature Biotechnology, 2006Co-Authors: Daniel Steiner, Patrik Forrer, Michael T Stumpp, Andreas PlückthunAbstract:Signal sequences directing cotranslational translocation expand the range of proteins amenable to Phage Display
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Signal sequences directing cotranslational translocation expand the range of proteins amenable to Phage Display
Nature Biotechnology, 2006Co-Authors: Daniel Steiner, Patrik Forrer, Michael T Stumpp, Andreas PlückthunAbstract:Even proteins that fold well in bacteria are frequently Displayed poorly on filamentous Phages. Low protein presentation on Phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in Phage assembly, we tested the Display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher Display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the Display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved Phage Display from tenfold enrichment to >1,000-fold enrichment per Phage Display selection round. We named this method 'SRP Phage Display' and envision broad applicability, especially when Displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.
