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

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dc.contributor.advisorBentley, William Een_US
dc.contributor.authorLewandowski, Angelaen_US
dc.contributor.departmentChemical Engineeringen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2005-02-02T06:46:19Z
dc.date.available2005-02-02T06:46:19Z
dc.date.issued2004-12-06en_US
dc.description.abstractFusion 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.en_US
dc.format.extent731828 bytes
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/1903/2105
dc.language.isoen_US
dc.subject.pqcontrolledEngineering, Chemicalen_US
dc.titleTyrosine-based "activatable pro-tag": enzyme-catalyzed protein capture and releaseen_US
dc.typeThesisen_US

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