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    Tyrosine-based "activatable pro-tag": enzyme-catalyzed protein capture and release

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    No. of downloads: 2709

    Date
    2004-12-06
    Author
    Lewandowski, Angela
    Advisor
    Bentley, William E
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    Abstract
    Fusion tags are widely used in the recovery and purification of recombinant proteins. We have investigated the capture and release of two fusion proteins from cell extracts using the aminopolysaccharide chitosan. We have fused to green fluorescent protein (GFP) and to S-ribosylhomocysteinase (LuxS) a "pro-tag" consisting of five tyrosine residues that are "activated" by tyrosinase-catalyzed conversion into reactive o-quinones. The o-quinones react with the amino groups of chitosan, resulting in the covalent conjugation of the fusion protein to chitosan. The fusion protein is captured from solution by precipitation of the protein-chitosan conjugate due to the decrease in solubility of chitosan at higher pH. Additionally, chitosan is used to "pre-precipitate" cell extract contaminants such as nucleic acids and phospholipids, and thus, crudely purify the fusion protein remaining in solution. Finally, we released the fusion protein from chitosan back into solution using the chitosan-hydrolyzing enzyme chitosanase as an alternative to protease cleavage.
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    http://hdl.handle.net/1903/2105
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    • Chemical and Biomolecular Engineering Theses and Dissertations
    • UMD Theses and Dissertations

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