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dc.contributor.advisorIverson, Brent L.en
dc.contributor.advisorMahal, Lara K.en
dc.creatorPropheter, Daniel Champlinen
dc.date.accessioned2011-10-13T18:24:29Zen
dc.date.available2011-10-13T18:24:29Zen
dc.date.issued2011-08en
dc.date.submittedAugust 2011en
dc.identifier.urihttp://hdl.handle.net/2152/ETD-UT-2011-08-4017en
dc.descriptiontexten
dc.description.abstractThe cell surface is enveloped with a myriad of carbohydrates that form complex matrices of oligosaccharides. Carbohydrate recognition plays crucial and varying roles in cellular trafficking, differentiation, and bacterial pathogenesis. Lectin microarray technology presents a unique platform for the high-throughput analysis of these structurally diverse classes of biopolymers. One significant hinderance of this technology has been the limitation imposed by the set of commercially available plant lectins used in the array. To enhance the reproducibility and scope of the lectin panel, our lab generated a small set of bacteria-derived recombinant lectins. This dissertation describes the unique advantages that recombinant lectins have over traditional plant-derived lectins. The recombinant lectins are expressed with a common fusion tag, glutathione-S-transferase (GST), which can be used as an immobilization handle on glutathione (GSH)-modified substrates. Although protein immobilization via fusion tags in a microarray format is not novel, our work demonstrates that protein activity through site-specific immobilization is enhanced when the protein is properly oriented. Although orientation enhanced the activity of our GST-tagged recombinant lectins, the GSH-surface modification precluded the printing of non-GST-tagged lectins, such as the traditional plant lectins, thus limiting the structural resolution of our arrays. To solve this issue, we developed a novel print technique which allows the one-step deposition and orientation of GST-tagged proteins in a microarray format. To expand our view of the glycome, we further adapt this method for the in situ orientation of unmodified IgG and IgM antibodies using GST-tagged antibody-binding proteins. Another advantage of recombinant lectins is in the ease of genomic manipulation, wherein we could tailor the binding domain to bind a different antigen. We demonstrate this by producing non-binding variants of the recombinant lectins to act as negative controls in our microarrays. Along with the non-binding variants, we developed a lectin displayed on the surface of phage. In the hopes generating more novel lectins, I will describe our current efforts of lectin evolution using phage-displayed GafD. By generating novel tools in lectin microarray technology, we enhance our understanding of the role of carbohydrates on a global scale.en
dc.format.mimetypeapplication/pdfen
dc.language.isoengen
dc.subjectProtein microarrayen
dc.subjectLectin microarrayen
dc.subjectGlycomicsen
dc.subjectAntibody microarrayen
dc.subjectBacterial lectinen
dc.titleAdvances in protein microarray technology for glycomic analysisen
dc.date.updated2011-10-13T18:24:44Zen
dc.identifier.slug2152/ETD-UT-2011-08-4017en
dc.contributor.committeeMemberSiegel, Dionicio R.en
dc.contributor.committeeMemberKeatinge-Clay, Adrianen
dc.contributor.committeeMemberFast, Walter L.en
dc.description.departmentChemistryen
dc.type.genrethesisen
thesis.degree.departmentChemistryen
thesis.degree.disciplineChemistryen
thesis.degree.grantorUniversity of Texas at Austinen
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen


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