Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies
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{"title"=>"Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies", "type"=>"journal", "authors"=>[{"first_name"=>"Valencio", "last_name"=>"Salema", "scopus_author_id"=>"35096610900"}, {"first_name"=>"Elvira", "last_name"=>"Marín", "scopus_author_id"=>"22035460900"}, {"first_name"=>"Rocio", "last_name"=>"Martínez-Arteaga", "scopus_author_id"=>"12778488300"}, {"first_name"=>"David", "last_name"=>"Ruano-Gallego", "scopus_author_id"=>"55786459300"}, {"first_name"=>"Sofía", "last_name"=>"Fraile", "scopus_author_id"=>"6508063810"}, {"first_name"=>"Yago", "last_name"=>"Margolles", "scopus_author_id"=>"55862272300"}, {"first_name"=>"Xema", "last_name"=>"Teira", "scopus_author_id"=>"55861997500"}, {"first_name"=>"Carlos", "last_name"=>"Gutierrez", "scopus_author_id"=>"7202545218"}, {"first_name"=>"Gustavo", "last_name"=>"Bodelón", "scopus_author_id"=>"6506050773"}, {"first_name"=>"Luis Ángel", "last_name"=>"Fernández", "scopus_author_id"=>"16238777700"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"369873412", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0075126", "scopus"=>"2-s2.0-84884523021", "pmid"=>"24086454", "sgr"=>"84884523021"}, "id"=>"bc771588-ba28-3ce8-8a15-0ee0833e2bed", "abstract"=>"Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM(EHEC)). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM(EHEC) binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM(EHEC) was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.", "link"=>"http://www.mendeley.com/research/selection-single-domain-antibodies-immune-libraries-displayed-surface-e-coli-cells-two-%CE%B2domains-oppo", "reader_count"=>65, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>2, "Researcher"=>18, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>17, "Student > Postgraduate"=>2, "Student > Master"=>11, "Other"=>3, "Student > Bachelor"=>3, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>2, "Researcher"=>18, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>17, "Student > Postgraduate"=>2, "Student > Master"=>11, "Other"=>3, "Student > Bachelor"=>3, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>5, "Biochemistry, Genetics and Molecular Biology"=>17, "Agricultural and Biological Sciences"=>30, "Medicine and Dentistry"=>4, "Neuroscience"=>1, "Arts and Humanities"=>1, "Sports and Recreations"=>1, "Chemical Engineering"=>1, "Chemistry"=>4, "Linguistics"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>4}, "Sports and Recreations"=>{"Sports and Recreations"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>30}, "Linguistics"=>{"Linguistics"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>17}, "Unspecified"=>{"Unspecified"=>5}, "Chemical Engineering"=>{"Chemical Engineering"=>1}, "Arts and Humanities"=>{"Arts and Humanities"=>1}}, "reader_count_by_country"=>{"Uruguay"=>1, "United States"=>1, "Brazil"=>1, "United Kingdom"=>1, "Switzerland"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1212535"], "description"=>"<p>(<b>A</b>) Scheme of EhaA autotransporter and VHHA fusions (left), showing N-terminal SP, secreted passenger or VHH domain, and C-terminal β-domain. Model of VHHA fusion in the OM (right), with N-terminal VHH domain exposed to the extracellular milieu and with C-EhaA β-barrel inserted in the OM. These domains are connected with the E-tag epitope and the internal α-helical linker of the β-barrel. (<b>B</b>) Scheme of Intimin and NVHH fusions (left), showing N-terminal SP, LysM and β-domains, and secreted D0-D3 Ig-like and lectin-like domains, or VHH domain replacing D1-D3 in NVHH fusions. Model of NVHH fusion in the OM (right), with N-terminal LysM domain in the periplasm, β-barrel with linker in the OM, and connecting with C-terminal D0 and VHH domains exposed to the extracellular milieu. The E-tag and myc-tag epitopes flanking the VHH domain are indicated. (<b>C</b>) and (<b>D</b>) Western blots of whole-cell protein extracts from induced <i>E. coli</i> UT5600 harbouring pVgfpA (C) or pNVgfp (D). Intact <i>E. coli</i> cells were incubated with (+) or without (-) the indicated protease, Trypsin or Proteinase-K (ProtK), before lysis. Protein extracts were prepared in SDS (C) or SDS-urea (D) sample buffers and boiled (+) or not boiled (-) before SDS-PAGE. Western blots were developed with anti-E or anti-myc mAb, as indicated. The positions of full-length VgfpA and NVgfp fusions are labeled with arrows. Asterisks indicate protein bands detected in protease-treated samples. The mass of protein markers (in kDa) is shown on the left.</p>", "links"=>[], "tags"=>["vhh", "sdabs", "ehaa", "intimin"], "article_id"=>805579, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_E_coli_cell_surface_display_of_VHH_sdAbs_with_EhaA_and_Intimin_domains_/805579", "title"=>"<i>E. coli</i> cell surface display of VHH sdAbs with EhaA and Intimin β-domains.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-23 01:59:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/1212536"], "description"=>"<p>Fluorescent flow cytometry analysis of induced <i>E. coli</i> UT5600 cells bearing the indicated plasmids: pAK-Not (control), pVgfpA, and pNVgfp. Histograms show the fluorescence intensity of bacteria stained with anti-E or anti-myc mAbs (as indicated) and secondary anti-mouse IgG-Alexa 488 (left panels) or incubated with biotinylated antigens (GFP or BSA, as labeled) and secondary Streptavidin-phycoerythrin (PE) (right panels).</p>", "links"=>[], "tags"=>["antigen", "binding", "vgfpa", "nvgfp"], "article_id"=>805580, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_E_coli_cell_surface_display_and_antigen_binding_activity_of_VgfpA_and_NVgfp_fusions_/805580", "title"=>"<i>E. coli</i> cell surface display and antigen binding activity of VgfpA and NVgfp fusions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-23 01:59:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/1212537"], "description"=>"<p>(<b>A</b>) Fluorescent flow cytometry analysis of induced <i>E. coli</i> EcM1 cells expressing VHHA or NVHH immune libraries anti-TirM<sub>EHEC</sub> (as indicated). Control cells carried the empty vector pAK-Not. Histograms show the fluorescence intensity of bacteria stained with anti-E or anti-myc mAbs (as labeled) and secondary anti-mouse IgG-Alexa 488. (<b>B</b>) Western blots of whole-cell protein extracts from induced <i>E. coli</i> EcM1 cells expressing VHHA or NVHH immune libraries anti-TirM<sub>EHEC</sub> (as indicated). Protein extracts were prepared in SDS (VHHA library) or SDS-urea (NVHH library) sample buffers and boiled (+) or not boiled (-) before SDS-PAGE. Western blots were developed with anti-E mAb. The positions of full-length fusions are labeled with arrows. The mass of protein markers (in kDa) is shown on the left.</p>", "links"=>[], "tags"=>["vhha", "nvhh"], "article_id"=>805581, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_E_coli_cell_surface_display_of_VHHA_and_NVHH_immune_libraries_/805581", "title"=>"<i>E. coli</i> cell surface display of VHHA and NVHH immune libraries.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-23 01:59:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/1212538"], "description"=>"<p>(<b>A</b>) General scheme summarizing the steps followed during MACS of an <i>E. coli</i> display library of sdAb with a biotinylated antigen. <i>E. coli</i> cells binding the biotinylated antigen are captured in a MACS column held in a magnet, while <i>E. coli</i> cells that do not bind the antigen are washed out of the column. Elution of bound bacteria is done with fresh LB media upon column removal from the magnet. The CFU in the Washed and Bound fractions are determined by plating. (<b>B</b>) Fluorescent flow cytometry analysis of IPTG-induced <i>E. coli</i> EcM1 cells expressing VHHA (top panel) or NVHH (bottom panel) immune libraries, or their respective sublibraries enriched after the indicated round of MACS with biotinylated TirM<sub>EHEC</sub>. Histograms show the fluorescence intensity of bacteria incubated with biotinylated TirM<sub>EHEC</sub> and secondary Streptavidin-PE.</p>", "links"=>[], "tags"=>["sorting", "libraries", "vhha", "nvhh", "biotinylated", "antigen"], "article_id"=>805582, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Magnetic_cell_sorting_of_E_coli_display_libraries_VHHA_and_NVHH_with_biotinylated_antigen_TirM_EHEC_/805582", "title"=>"Magnetic cell sorting of <i>E. coli</i> display libraries VHHA and NVHH with biotinylated antigen TirM<sub>EHEC</sub>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-23 01:59:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/1212540"], "description"=>"<p>Fluorescent flow cytometry analysis of induced <i>E. coli</i> EcM1 cells bearing the indicated plasmids selected from (<b>A</b>) the VHHA library: pVTIR1A, pVTIR2A, pVTIR3A; and (<b>B</b>) from the NVHH library: pNVTIR1, pNVTIR4, pNVTIR5. Histograms show the fluorescence intensity of bacteria incubated with biotinylated antigens (TirM<sub>EHEC</sub> or BSA, as labeled) and secondary Streptavidin-PE.</p>", "links"=>[], "tags"=>["cells", "displaying", "clones", "vhha", "nvhh"], "article_id"=>805584, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Binding_to_TirM_EHEC_by_E_coli_cells_displaying_selected_clones_from_VHHA_and_NVHH_libraries_/805584", "title"=>"Binding to TirM<sub>EHEC</sub> by <i>E. coli</i> cells displaying selected clones from VHHA and NVHH libraries.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-23 01:59:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/1212541"], "description"=>"<p>ELISA against TirM<sub>EHEC</sub> of sdAbs secreted into culture media as E-tagged HlyA fusions from the indicated VTIR clones and one a negative control (Vamy) [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075126#B35\" target=\"_blank\">35</a>]. The plot shows the average OD values at 490 nm with standard error from duplicate experimental samples obtained with the secreted sdAbs at the indicated concentrations. ELISA were developed with anti-E-tag mAb and anti-mouse-POD. ELISA signals against a control antigen (BSA) are subtracted from the represented values.</p>", "links"=>[], "tags"=>["sdabs"], "article_id"=>805585, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ELISA_of_sdAbs_selected_by_E_coli_display_against_TirM_EHEC_/805585", "title"=>"ELISA of sdAbs selected by <i>E. coli</i> display against TirM<sub>EHEC</sub>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-23 01:59:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/1212542"], "description"=>"<p>(<b>A</b>) SPR sensograms monitoring real-time association and dissociation of purified sdAb VTIR1 (at the indicated concentrations) to biotinylated TirM<sub>EHEC</sub> immobilized onto a Streptavidin-SA sensor chip. The increase in resonance units (RU) is recorded along time (in seconds). Dissociation of VTIR1 is evaluated by injection of buffer at the time indicated with an arrow. (<b>B</b>) RU values at 220 seconds (labeled with a rectangle in A) are plotted <i>versus</i> the different concentrations of VTIR1. The curve was fitted by non-linear least squares regression. (<b>C</b>) The K<sub>D</sub> of VTIR1 was estimated by flow cytometry analysis of <i>E. coli</i> cells expressing NVTIR1 incubated with different concentrations of biotinylated TirM<sub>EHEC</sub> (1-20 nM) under equilibrium conditions. The mean fluorescent intensities (MFI) of bacteria, after labeling with Streptavidin-PE, were plotted <i>versus</i> the concentration of TirM<sub>EHEC</sub> used in the assays. The curve was fitted by non-linear least squares regression.</p>", "links"=>[], "tags"=>["equilibrium", "dissociation", "sdab", "vtir1", "spr"], "article_id"=>805586, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Determination_of_the_equilibrium_dissociation_constant_K_D_of_sdAb_VTIR1_by_SPR_and_E_coli_display_/805586", "title"=>"Determination of the equilibrium dissociation constant (K<sub>D</sub>) of sdAb VTIR1 by SPR and <i>E. coli</i> display.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-23 01:59:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/1212544", "https://ndownloader.figshare.com/files/1212545", "https://ndownloader.figshare.com/files/1212546", "https://ndownloader.figshare.com/files/1212547", "https://ndownloader.figshare.com/files/1212548", "https://ndownloader.figshare.com/files/1212549", "https://ndownloader.figshare.com/files/1212550"], "description"=>"<div><p>Screening of antibody (Ab) libraries by direct display on the surface of <i>E. coli</i> cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic <i>E. coli</i> O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or V<sub>HH</sub>) on the surface of <i>E. coli</i> K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM<sub>EHEC</sub>). We demonstrated that both systems displayed functional sdAbs on the surface of <i>E. coli</i> cells with little proteolysis and cellular toxicity, although <i>E. coli</i> cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both <i>E. coli</i> display libraries were screened for TirM<sub>EHEC</sub> binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM<sub>EHEC</sub> was demonstrated by flow cytometry of <i>E. coli</i> cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the <i>E. coli</i> cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.</p> </div>", "links"=>[], "tags"=>["antibodies", "libraries", "displayed", "cells", "topologies"], "article_id"=>805588, "categories"=>["Biological Sciences"], "users"=>["Valencio Salema", "Elvira Marín", "Rocio Martínez-Arteaga", "David Ruano-Gallego", "Sofia Fraile", "Yago Margolles", "Xema Teira", "Carlos Gutierrez", "Gustavo Bodelón", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075126.s001", "https://dx.doi.org/10.1371/journal.pone.0075126.s002", "https://dx.doi.org/10.1371/journal.pone.0075126.s003", "https://dx.doi.org/10.1371/journal.pone.0075126.s004", "https://dx.doi.org/10.1371/journal.pone.0075126.s005", "https://dx.doi.org/10.1371/journal.pone.0075126.s006", "https://dx.doi.org/10.1371/journal.pone.0075126.s007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Selection_of_Single_Domain_Antibodies_from_Immune_Libraries_Displayed_on_the_Surface_of_E_coli_Cells_with_Two_Domains_of_Opposite_Topologies/805588", "title"=>"Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of <i>E. coli</i> Cells with Two β-Domains of Opposite Topologies", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-09-23 01:59:13"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Physical sciences", "average_usage"=>[254, 431, 547, 651, 748, 842, 932, 1017, 1098, 1178, 1259, 1336, 1404]}, {"subject_area"=>"/Physical sciences/Materials science", "average_usage"=>[250, 417, 535, 649, 758, 857, 952, 1036, 1113, 1206, 1287, 1353, 1429]}]}
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