Method Development for Efficient Incorporation of Unnatural Amino Acids

Handle URI:
http://hdl.handle.net/10754/315826
Title:
Method Development for Efficient Incorporation of Unnatural Amino Acids
Authors:
Harris, Paul D.
Abstract:
The 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.
Advisors:
Eppinger, Jörg ( 0000-0001-7886-7059 )
Committee Member:
Hamdan, Samir; Khashab, Niveen M.
KAUST Department:
Physical Sciences and Engineering (PSE) Division
Program:
Chemical Sciences
Issue Date:
Apr-2014
Type:
Thesis
Appears in Collections:
Theses; Physical Sciences and Engineering (PSE) Division; Chemical Science Program

Full metadata record

DC FieldValue Language
dc.contributor.advisorEppinger, Jörgen
dc.contributor.authorHarris, Paul D.en
dc.date.accessioned2014-04-14T11:20:21Z-
dc.date.available2014-04-14T11:20:21Z-
dc.date.issued2014-04en
dc.identifier.urihttp://hdl.handle.net/10754/315826en
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.en
dc.language.isoenen
dc.subjectunnatural amino aciden
dc.subjectfluorescent proteinen
dc.subjectprotein translationen
dc.subjectfluorescent labelingen
dc.subjectprotein expressionen
dc.subjecthigh density cell cultureen
dc.titleMethod Development for Efficient Incorporation of Unnatural Amino Acidsen
dc.typeThesisen
dc.contributor.departmentPhysical Sciences and Engineering (PSE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberHamdan, Samiren
dc.contributor.committeememberKhashab, Niveen M.en
thesis.degree.disciplineChemical Sciencesen
thesis.degree.nameMaster of Scienceen
dc.person.id124300en
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