Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

Scientific Reports
Debra T HansenKathryn Sykes

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.

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Citations

Oct 4, 2019·Scientific Reports·Luhui ShenStephen Albert Johnston
Sep 21, 2017·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Aysha S AnsariHasan Uludağ

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Datasets Mentioned

BETA
GM094599

Methods Mentioned

BETA
electron cryomicroscopy
ELISA
transfection
PCR
electrophoresis
Chips
enzyme-linked immunosorbent assays

Software Mentioned

TMHMM
SOSUI
TMPred
TopPred
SignalP
OMEP

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Antibodies produced by B cells are highly specific for antigen as a result of random gene recombination and somatic hypermutation and affinity maturation. As the main effector of the humoral immune system, antibodies can neutralize foreign cells. Find the latest research on antibody specificity here.

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