Development of a novel nanoparticle bioconjugation strategy utilizing coiled-coil protein domains

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dc.contributor Bao, Yuping
dc.contributor Jenny, Matthew J.
dc.contributor LeBlanc, Paul A.
dc.contributor O'Donnell, Janis M.
dc.contributor.advisor Duffy, Carol Hill, Brandon 2017-03-02T19:55:19Z 2017-03-02T19:55:19Z 2016
dc.identifier.other u0015_0000001_0002544
dc.identifier.other Hill_alatus_0004D_12915
dc.description Electronic Thesis or Dissertation
dc.description.abstract Nanotechnology has played a prominent role in the rapid development of technological advances used for biomedical applications. Such advances explore the role of nanoparticles (NPs) in 1) MRI contrasting/cell labeling, 2) targeted drug delivery, and 3) intravenous dosing for therapeutic and or diagnostic purposes. The expanded use of nanotechnology in medicine requires the development of optimal NP bioconjugation strategies. The technologies currently utilized for attachment of biological molecules to inorganic NP surfaces include 1-ethyl-3-(3 dimethylaminopropyl carbodimide hydrochloride-N-hydroxysulfosuccinimide (EDC-NHS) conjugation, catechol conjugation, and biotin-steptavadin conjugation. There are inherent drawbacks with each of these strategies as they do not allow for directional attachment of the biological molecule and/or they require chemical conditions that damage the structure/function of the biological molecule. To address this issue, we developed an E/K coiled-coil mechanism for conjugation of peptides and proteins to iron oxide nanoparticle (IONP) surfaces. I utilized monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (EGFP) as model proteins for the proof-of-concept studies described in this dissertation. First, I determined optimal conditions to promote expression and purification of the E-coil fusion protein. I was ultimately able to obtain yields of 1 mg/liter culture for this small (~5.8 kDa) protein. Second, I determined optimal conditions to promote expression and purification of the mRFP-K-coil and EGFP-K-coil fusion proteins. For the proof-of-concept studies, I focused on determining whether the coiled-coil approach could be utilized to directionally attach large proteins (mRFP and EGFP) to IONP surfaces while maintaining their complex tertiary structures. Although, we did not observe fluorescence following coiled-coil conjugation of mRFP or EGFP to IONPs, likely due to quenching by the iron oxide core, fluorescence was maintained upon coiled-coil conjugation to an E-coil-coated cobalt resin. The tertiary structures of mRFP and EGFP are essential to their fluorescence. Thus, these studies demonstrated a critical advantage of the coiled-coil conjugation strategy in maintaining the structure/function of the conjugated protein. Finally, I demonstrated that multiple, different proteins can be attached to IONPs; a feature vital to the development of a multifunctional NP platform. The coiled-coil NP bioconjugation strategy described in this dissertation can be utilized in future studies to expand the use of inorganic NPs in biomedical applications.
dc.format.extent 117 p.
dc.format.medium electronic
dc.format.mimetype application/pdf
dc.language English
dc.language.iso en_US
dc.publisher University of Alabama Libraries
dc.relation.ispartof The University of Alabama Electronic Theses and Dissertations
dc.relation.ispartof The University of Alabama Libraries Digital Collections
dc.relation.hasversion born digital
dc.rights All rights reserved by the author unless otherwise indicated.
dc.subject.other Nanoscience
dc.subject.other Nanotechnology
dc.title Development of a novel nanoparticle bioconjugation strategy utilizing coiled-coil protein domains
dc.type thesis
dc.type text University of Alabama. Dept. of Biological Sciences Biological Sciences The University of Alabama doctoral Ph.D.

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