UMD Theses and Dissertations

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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.

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Author: search.filters.author.Rozak, David AnthonySubject: search.filters.subject.albumin binding domain

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    Using Recombinant PCR to Study Sequence Polymorphisms in a Family of Compact Albumin Binding Domains
    (2005-11-14) Rozak, David Anthony; Bryan, Philip N; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Sixteen 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.