@article{ea6065254d814593886c74bb601140cf,
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.",
author = "Debra Hansen and Robida, {Mark D.} and Craciunescu, {Felicia M.} and Loskutov, {Andrey V.} and Katerina D{\"o}rner and Rodenberry, {John Charles} and Xiao Wang and Olson, {Tien L.} and Hetal Patel and Petra Fromme and Sykes, {Kathryn F.}",
note = "Funding Information: Membrane protein targets. Membrane proteins in this study (Supplementary Table 1) were selected by the Center for Membrane Proteins in Infectious Diseases (MPID; grant GM094599), as part of the PSI:Biology program under the U.S. National Institutes of Health{\textquoteright}s Protein Structure Initiative. Information on MPID targets is available at the Structural Biology Knowledgebase Technology Portal51 (http://sbkb.org). Due to technical difficulties that are commonly encountered when working with membrane proteins, the methods are presented here in necessary detail. Funding Information: We thank Stephen A. Johnston for support and helpful discussions; Kelli L. Trimble, Vasiliy A. Loskutov, Farzana Sarder, Tracie M. Smith, and Steven M. Montoya for preparation of materials; Deborah Luzader for fluorescence analyses; Kristen Seifert and Alexandre Y. Borovkov for cloning contributions; Danielle Lussier for technical assistance with micronanoplexes; Kurt Whittemore for assistance with mice; and Jeffrey L. Hansen for the use of pCDF-BAD. This work was supported by a U.S. National Institutes of Health grant GM094599 (P.F. and K.F.S.).",
year = "2016",
month = feb,
day = "24",
doi = "10.1038/srep21925",
language = "English (US)",
volume = "6",
journal = "Scientific reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
}