The Experts below are selected from a list of 18903 Experts worldwide ranked by ideXlab platform

Johan Rockberg - One of the best experts on this subject based on the ideXlab platform.

  • SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping
    Nucleic acids research, 2019
    Co-Authors: Francis Jingxin Hu, Magnus Lundqvist, Mathias Uhlén, Johan Rockberg
    Abstract:

    Mutagenesis libraries are essential for combinato-rial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonu-cleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv an-tibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with Bacterial Display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • Combination of phage and Gram-positive Bacterial Display of human antibody repertoires enables isolation of functional high affinity binders
    New Biotechnology, 2018
    Co-Authors: Francis Jingxin Hu, Anna Luisa Volk, Anna Säll, Carl Borrebaeck, Mathias Uhlén, Helena Persson, Johan Rockberg
    Abstract:

    Surface Display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various Display systems available, phage Display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive Bacterial host for Display of a library of human antibody genes which, when combined with phage Display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive Bacterial Display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

  • phage and gram positive Bacterial Display of human antibody repertoires enables isolation of functional high affinity binders
    New Biotechnology, 2017
    Co-Authors: Anna Luisa Volk, Anna Säll, Carl Borrebaeck, Mathias Uhlén, Helena Persson, Johan Rockberg
    Abstract:

    Surface Display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various Display systems, pha ...

  • proteome wide epitope mapping of antibodies using ultra dense peptide arrays
    Molecular & Cellular Proteomics, 2014
    Co-Authors: Bjorn Forsstrom, Barbara Bislawska Axnas, Klauspeter Stengele, Jochen Buhler, Francis Jingxin Hu, Elton P Hudson, Todd Richmond, Thomas J Albert, Peter Nilsson, Johan Rockberg
    Abstract:

    Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy.This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on Bacterial Display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry.Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments.

Patrick S Daugherty - One of the best experts on this subject based on the ideXlab platform.

  • Identification of disease-specific motifs in the antibody specificity repertoire via next-generation sequencing
    Scientific Reports, 2016
    Co-Authors: Robert J Pantazes, Joel Bozekowski, Kelly N. Ibsen, Jack Reifert, Joseph A Murray, Patrick S Daugherty
    Abstract:

    Disease-specific antibodies can serve as highly effective biomarkers but have been identified for only a relatively small number of autoimmune diseases. A method was developed to identify disease-specific binding motifs through integration of Bacterial Display peptide library screening, next-generation sequencing (NGS) and computational analysis. Antibody specificity repertoires were determined by identifying bound peptide library members for each specimen using cell sorting and performing NGS. A computational algorithm, termed Identifying Motifs Using Next-generation sequencing Experiments (IMUNE), was developed and applied to discover disease-and healthy control-specific motifs. IMUNE performs comprehensive pattern searches, identifies patterns statistically enriched in the disease or control groups and clusters the patterns to generate motifs. Using celiac disease sera as a discovery set, IMUNE identified a consensus motif (QPEQPF[PS]E) with high diagnostic sensitivity and specificity in a validation sera set, in addition to novel motifs. Peptide Display and sequencing (Display-Seq) coupled with IMUNE analysis may thus be useful to characterize antibody repertoires and identify disease-specific antibody epitopes and biomarkers.

  • persistence of elevated deamidated gliadin peptide antibodies on a gluten free diet indicates nonresponsive coeliac disease
    Alimentary Pharmacology & Therapeutics, 2014
    Co-Authors: Bradley N Spatola, Katri Kaukinen, Pekka Collin, Markku Maki, Martin F Kagnoff, Patrick S Daugherty
    Abstract:

    Summary Background Histologically nonresponsive coeliac disease (NRCD) is a potentially serious condition diagnosed during the follow-up of coeliac disease (CD) when patients have persistent villous atrophy despite following a gluten-free diet (GFD). Aim As current assessments of recovery are limited to invasive and costly serial duodenal biopsies, we sought to identify antibody biomarkers for CD patients that do not respond to traditional therapy. Methods Bacterial Display peptide libraries were screened by flow cytometry to identify epitopes specifically recognised by antibodies from patients with NRCD, but not by antibodies from responsive CD patients. Deamidated gliadin was confirmed to be the antigen mimicked by library peptides using ELISA with sera from NRCD (n = 15) and responsive CD (n = 45) patients on a strict GFD for at least 1 year. Results The dominant consensus epitope sequence identified by unbiased library screening QPxx(A/P)FP(E/D) was highly similar to reported deamidated gliadin peptide (dGP) B-cell epitopes. Measurement of anti-dGP IgG titre by ELISA discriminated between NRCD and responsive CD patients with 87% sensitivity and 89% specificity. Importantly, dGP antibody titre correlated with the severity of mucosal damage indicating that IgG dGP titres may be useful to monitor small intestinal mucosal recovery on a GFD. Conclusions The finding of increased levels of anti-dGP IgG antibodies in CD patients on strict GFDs effectively identifies patients with NRCD. Finally, anti-dGP IgG assays may be useful to monitor mucosal damage and histological improvement in CD patients on a strict GFD.

  • Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro
    Biological Chemistry, 2014
    Co-Authors: Tyler D. Shropshire, Sridharan Rajagopalan, Jack Reifert, Stuart C. Feinstein, David Baker, Patrick S Daugherty
    Abstract:

    The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a Bacterial Display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)↓(Y/F)↓(S/A/G/T) or RXΦ(Y/F)↓(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K 16 L 17 V 18 F 19 F 20 A 21 ) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ 42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ 42 -mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.

  • Design of a cyclotide antagonist of neuropilin-1 and -2 that potently inhibits endothelial cell migration.
    ACS chemical biology, 2013
    Co-Authors: Jennifer A. Getz, Olivier Cheneval, David J. Craik, Patrick S Daugherty
    Abstract:

    Neuropilin-1 and -2 are critical regulators of angiogenesis, lymphangiogenesis, and cell survival as receptors for multiple growth factors. Disulfide-rich peptides that antagonize the growth factor receptors neuropilin-1 and neuropilin-2 were developed using Bacterial Display libraries. Peptide ligands specific for the VEGFA binding site on neuropilin-1 were identified by screening a library of disulfide-rich peptides derived from the thermostable, protease-resistant cyclotide kalata B1. First generation ligands were subjected to one cycle of affinity maturation to yield acyclic peptides with affinities of 40-60 nM and slow dissociation rate constants (∼1 × 10(-3) s(-1)). Peptides exhibited equivalent affinities for human and mouse neuropilin-1 and cross-reacted with human neuropilin-2 with lower affinity. A C-to-N cyclized variant (cyclotide) of one neuropilin ligand retained high affinity, exhibited increased protease resistance, and conferred improved potency for inhibiting endothelial cell migration in vitro (EC50 ≈ 100 nM). These results demonstrate that potent, target-specific cyclotides can be created by evolutionary design and that backbone cyclization can confer improved pharmacological properties.

  • Bacterial Display using circularly permuted outer membrane protein ompx yields high affinity peptide ligands
    Protein Science, 2006
    Co-Authors: Jeffrey J. Rice, Paul H. Bessette, Aaron Schohn, Kevin T Boulware, Patrick S Daugherty
    Abstract:

    A Bacterial Display methodology was developed for N- and C-terminal Display and demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. To overcome limitations of insertional fusion Display libraries, a new scaffold was developed through circular permutation of the Escherichia coli outer membrane protein OmpX that presents both N and C termini on the external cell surface. Circularly permuted OmpX (CPX) Display was directly compared to insertional fusion Display by screening comparable peptide libraries in each format using magnetic and fluorescence activated cell sorting. CPX Display enabled in situ measurement of dissociation rate constants with improved accuracy and, consequently, improved affinity discrimination during screening and ranking of isolated clones. Using streptavidin as a model target, Bacterial Display yielded the well-characterized HPQ/M motif obtained previously using several alternative peptide Display systems, as well as three additional motifs (LI/V CQNVCY, CGWMYF/YxEC, ERCWYVMHWPCNA). Using CPX Display, a very high affinity streptavidin-binding peptide was isolated having a dissociation rate constant koff = 0.002sec-1 even after grafting to the C terminus of an unrelated protein. Comparison of individual clones obtained from insertional fusion and terminal fusion libraries suggests that the N-terminal Display yields sequences with greater diversity, affinity, and modularity. CPX Bacterial Display thus provides a highly effective method for screening peptide libraries to rapidly generate ligands with high affinity and specificity.

Francis Jingxin Hu - One of the best experts on this subject based on the ideXlab platform.

  • SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping
    Nucleic acids research, 2019
    Co-Authors: Francis Jingxin Hu, Magnus Lundqvist, Mathias Uhlén, Johan Rockberg
    Abstract:

    Mutagenesis libraries are essential for combinato-rial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonu-cleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv an-tibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with Bacterial Display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • Combination of phage and Gram-positive Bacterial Display of human antibody repertoires enables isolation of functional high affinity binders
    New Biotechnology, 2018
    Co-Authors: Francis Jingxin Hu, Anna Luisa Volk, Anna Säll, Carl Borrebaeck, Mathias Uhlén, Helena Persson, Johan Rockberg
    Abstract:

    Surface Display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various Display systems available, phage Display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive Bacterial host for Display of a library of human antibody genes which, when combined with phage Display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive Bacterial Display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

  • proteome wide epitope mapping of antibodies using ultra dense peptide arrays
    Molecular & Cellular Proteomics, 2014
    Co-Authors: Bjorn Forsstrom, Barbara Bislawska Axnas, Klauspeter Stengele, Jochen Buhler, Francis Jingxin Hu, Elton P Hudson, Todd Richmond, Thomas J Albert, Peter Nilsson, Johan Rockberg
    Abstract:

    Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy.This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on Bacterial Display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry.Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments.

Mathias Uhlén - One of the best experts on this subject based on the ideXlab platform.

  • SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping
    Nucleic acids research, 2019
    Co-Authors: Francis Jingxin Hu, Magnus Lundqvist, Mathias Uhlén, Johan Rockberg
    Abstract:

    Mutagenesis libraries are essential for combinato-rial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonu-cleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv an-tibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with Bacterial Display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • Combination of phage and Gram-positive Bacterial Display of human antibody repertoires enables isolation of functional high affinity binders
    New Biotechnology, 2018
    Co-Authors: Francis Jingxin Hu, Anna Luisa Volk, Anna Säll, Carl Borrebaeck, Mathias Uhlén, Helena Persson, Johan Rockberg
    Abstract:

    Surface Display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various Display systems available, phage Display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive Bacterial host for Display of a library of human antibody genes which, when combined with phage Display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive Bacterial Display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

  • phage and gram positive Bacterial Display of human antibody repertoires enables isolation of functional high affinity binders
    New Biotechnology, 2017
    Co-Authors: Anna Luisa Volk, Anna Säll, Carl Borrebaeck, Mathias Uhlén, Helena Persson, Johan Rockberg
    Abstract:

    Surface Display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various Display systems, pha ...

Anna Luisa Volk - One of the best experts on this subject based on the ideXlab platform.

  • Combination of phage and Gram-positive Bacterial Display of human antibody repertoires enables isolation of functional high affinity binders
    New Biotechnology, 2018
    Co-Authors: Francis Jingxin Hu, Anna Luisa Volk, Anna Säll, Carl Borrebaeck, Mathias Uhlén, Helena Persson, Johan Rockberg
    Abstract:

    Surface Display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various Display systems available, phage Display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive Bacterial host for Display of a library of human antibody genes which, when combined with phage Display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive Bacterial Display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

  • phage and gram positive Bacterial Display of human antibody repertoires enables isolation of functional high affinity binders
    New Biotechnology, 2017
    Co-Authors: Anna Luisa Volk, Anna Säll, Carl Borrebaeck, Mathias Uhlén, Helena Persson, Johan Rockberg
    Abstract:

    Surface Display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various Display systems, pha ...