Using Recombinant PCR to Study Sequence Polymorphisms in a Family of Compact Albumin Binding Domains

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dc.contributor.advisorBryan, Philip Nen_US
dc.contributor.authorRozak, David Anthonyen_US
dc.contributor.departmentMolecular and Cell Biologyen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2006-02-04T06:52:27Z
dc.date.available2006-02-04T06:52:27Z
dc.date.issued2005-11-14en_US
dc.description.abstractSixteen homologs of a compact albumin binding domain were previously identified in six proteins and four bacterial species. These domains, which exhibit varied affinities for different albumins, have been shown to support bacterial growth in vitro, and may contribute to host specificity. This dissertation describes the development of a robust PCR-based recombination technique, which is applied to representatives of the albumin binding domain to identify and understand the impact of sequence polymorphisms on domain stability and function. Analysis of phage-selected recombinants highlights the potential impact of multiple mutations in stabilizing the selected domains and improving albumin binding through gains in hydrophilic surface area, direct modifications to the binding interface, and subtle changes in the position of the third helix. The most common mutant was encoded by three fourths of the selected phage and exhibited 5 and 10 fold increases in human and guinea pig albumin binding constants compared to the wild type streptococcal domain (G148-GA3). This study serves to validate further the application of in vitro recombination and phage display in the analysis of sequence polymorphisms. The recombination technique itself is shown to be well suited for producing multiple recombination events among compact heterologous domains and appears to offer several advantages over traditional DNA shuffling techniques.en_US
dc.format.extent3541931 bytes
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/1903/3099
dc.language.isoen_US
dc.subject.pqcontrolledBiology, Molecularen_US
dc.subject.pquncontrolledalbumin binding domainen_US
dc.subject.pquncontrolledin vitro recombinationen_US
dc.subject.pquncontrolledphage displayen_US
dc.subject.pquncontrolledthermodynamicsen_US
dc.titleUsing Recombinant PCR to Study Sequence Polymorphisms in a Family of Compact Albumin Binding Domainsen_US
dc.typeDissertationen_US
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