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.

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain

“…This method allows the transfer of a foreign gene quickly and reliably. It can also be used to produce an antibody specifically against a protein without many purification steps [24]. We previously reported that antibodies produced by genetic immunization using human nephrin DNA had high specificity [25,26].…”

Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

“…We recently reported the first description of the efficiency of any DNA immunization approach specific for membrane proteins (Hansen et al, 2016). We applied the DNA-gold micronanoplex method (Svarovsky et al, 2009) and the immunization vector pCMVi-LSrCOMPTT (Table 1; Figure 1) to a structurally diverse set of 17 membrane proteins from two Biosafety Level 3 pathogens, and demonstrated polyclonal antibody production for more than half of the targets (Hansen et al, 2016).…”

“…We applied the DNA-gold micronanoplex method (Svarovsky et al, 2009) and the immunization vector pCMVi-LSrCOMPTT (Table 1; Figure 1) to a structurally diverse set of 17 membrane proteins from two Biosafety Level 3 pathogens, and demonstrated polyclonal antibody production for more than half of the targets (Hansen et al, 2016). A major validation of this study was that antibody production correlated with prior reports of target immunogenicity in infected or immunized hosts (Hansen et al, 2016). To support widespread use, all plasmid vectors from this study have been made available through the non-profit DNASU Plasmid Repository (Seiler et al, 2014).…”

“…Our recent report (Hansen et al, 2016) included pCMVi-LS-LTA-R192G and pCMVi-LS-LTB (Table 1), which encode mouse-codon-optimized sequences of the enterotoxigenic E. coli heat-labile enterotoxin subunits A (inactive mutant R192G) and B that are fused to the α 1 -antitrypsin leader sequence (Whitlock et al, 2011). These vectors have also been applied to vaccine development (Whitlock et al, 2011); however, the effect of these vectors on antibody titers has not been specifically evaluated (Whitlock et al, 2011; Hansen et al, 2016). For soluble (i.e., non-transmembrane) proteins, inclusion of the adjuvant vectors pCMVi-mGMCSF and pCMVi-Flt3L (Table 1) led to improved titers (Chambers and Johnston, 2003).…”

“…CpGs can be included with plasmid DNA during incorporation into bullets (Hansen et al, 2016). CpG oligodeoxynucleotides with complete phosphorothioate linkages, which resist nuclease digestion, can be purchased (e.g., Invitrogen or Sigma) in desalted form and without further purification.…”