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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Antibodies / isolation & purification*
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Antibodies / metabolism
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Antibody Specificity
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Antigens, Bacterial / genetics
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Antigens, Bacterial / immunology
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Bacterial Outer Membrane Proteins / genetics
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Bacterial Outer Membrane Proteins / immunology*
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Biolistics
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DNA, Bacterial / genetics
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DNA, Bacterial / immunology*
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Female
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Francisella tularensis / genetics
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Francisella tularensis / immunology*
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Francisella tularensis / pathogenicity
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Gene Expression Regulation
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Genetic Vectors / administration & dosage
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Gold / chemistry
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / immunology
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Immunization / instrumentation
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Immunization / methods
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Immunoconjugates / administration & dosage*
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Immunoconjugates / genetics
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Magnetite Nanoparticles / chemistry
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Mice
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Mice, Inbred BALB C
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Protein Biosynthesis
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / immunology
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Tularemia / immunology
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Tularemia / microbiology
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Tularemia / prevention & control*
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Virulence Factors / genetics
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Virulence Factors / immunology*
Substances
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Antibodies
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Antigens, Bacterial
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Bacterial Outer Membrane Proteins
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DNA, Bacterial
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FopA protein, Francisella tularensis
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Immunoconjugates
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Magnetite Nanoparticles
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Recombinant Fusion Proteins
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Virulence Factors
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Green Fluorescent Proteins
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Gold