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dc.contributor.advisorEppinger, Jörg
dc.contributor.authorHarris, Paul D.
dc.date.accessioned2014-04-14T11:20:21Z
dc.date.available2014-04-14T11:20:21Z
dc.date.issued2014-04
dc.identifier.doi10.25781/KAUST-1ZS6C
dc.identifier.urihttp://hdl.handle.net/10754/315826
dc.description.abstractThe synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.
dc.language.isoen
dc.subjectunnatural amino acid
dc.subjectfluorescent protein
dc.subjectprotein translation
dc.subjectfluorescent labeling
dc.subjectprotein expression
dc.subjecthigh density cell culture
dc.titleMethod Development for Efficient Incorporation of Unnatural Amino Acids
dc.typeThesis
dc.contributor.departmentPhysical Science and Engineering (PSE) Division
thesis.degree.grantorKing Abdullah University of Science and Technology
dc.contributor.committeememberHamdan, Samir
dc.contributor.committeememberKhashab, Niveen M.
thesis.degree.disciplineChemical Science
thesis.degree.nameMaster of Science
refterms.dateFOA2018-06-14T06:42:13Z


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