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dc.contributor.advisorArcus, Vickery L.
dc.contributor.authorSteemson, John Durand
dc.date.accessioned2011-08-14T21:35:46Z
dc.date.available2012-11-11T23:27:48Z
dc.date.issued2011
dc.identifier.citationSteemson, J. D. (2011). Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent (Thesis, Doctor of Philosophy (PhD)). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/5576en
dc.identifier.urihttps://hdl.handle.net/10289/5576
dc.description.abstractInteractions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies.
dc.format.mimetypeapplication/pdf
dc.format.mimetypeapplication/zip
dc.language.isoen
dc.publisherUniversity of Waikato
dc.rightsAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectOB-fold
dc.subjectdirected evolution
dc.subjectPhage display
dc.subjectprotien engineering
dc.titleDirected evolution and structural analysis of an OB-fold domain towards a specifc binding reagenten
dc.typeThesis
thesis.degree.grantorUniversity of Waikato
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (PhD)
dc.date.updated2011-08-10T01:29:16Z
pubs.place-of-publicationHamilton, New Zealanden_NZ


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