title: Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization.
abstract: Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.|Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.|School of Molecular Sciences (formerly the Department of Chemistry and Biochemistry), Arizona State University, Tempe, Arizona, USA.|U.S. National Institute of General Medical Sciences PSI:Biology Center for Membrane Proteins in Infectious Diseases.
authors.forename: Debra T
authors.initials: DT
authors.lastname: Hansen
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.|U.S. National Institute of General Medical Sciences PSI:Biology Center for Membrane Proteins in Infectious Diseases.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Robida
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.|Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.|U.S. National Institute of General Medical Sciences PSI:Biology Center for Membrane Proteins in Infectious Diseases.
authors.forename: Felicia M
authors.initials: FM
authors.lastname: Craciunescu
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Loskutov
authors.affiliation: School of Molecular Sciences (formerly the Department of Chemistry and Biochemistry), Arizona State University, Tempe, Arizona, USA.|U.S. National Institute of General Medical Sciences PSI:Biology Center for Membrane Proteins in Infectious Diseases.
authors.forename: Katerina
authors.initials: K
authors.lastname: Dörner
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: John-Charles
authors.initials: JC
authors.lastname: Rodenberry
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: Tien L
authors.initials: TL
authors.lastname: Olson
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: Hetal
authors.initials: H
authors.lastname: Patel
authors.affiliation: Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.|School of Molecular Sciences (formerly the Department of Chemistry and Biochemistry), Arizona State University, Tempe, Arizona, USA.|U.S. National Institute of General Medical Sciences PSI:Biology Center for Membrane Proteins in Infectious Diseases.
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
authors.affiliation: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.|U.S. National Institute of General Medical Sciences PSI:Biology Center for Membrane Proteins in Infectious Diseases.
authors.forename: Kathryn F
authors.initials: KF
authors.lastname: Sykes
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification* / Q000378:metabolism; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D019470:Biolistics; D004269:DNA, Bacterial / Q000235:genetics / Q000276:immunology*; D005260:Female; D005604:Francisella tularensis / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology; D007114:Immunization / Q000295:instrumentation / Q000379:methods; D018796:Immunoconjugates / Q000008:administration & dosage* / Q000235:genetics; D058185:Magnetite Nanoparticles / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014406:Tularemia / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology*
chemical_list: D000906:Antibodies; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; C083436:FopA protein, Francisella tularensis; D018796:Immunoconjugates; D058185:Magnetite Nanoparticles; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D049452:Green Fluorescent Proteins; D006046:Gold