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Genetic Code Expansion for Protein Activity Control and Protein Conjugation

Liu, Jihe (2018) Genetic Code Expansion for Protein Activity Control and Protein Conjugation. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Expanding the genetic code by unnatural amino acid (UAA) incorporation allows for control of protein structure and function in cells and animals. Genetic incorporations of several UAAs, including dithiolane lysine, azidoethyl and azidomethyl lysine, photocaged tyrosine/thiotyrosine/azatorysine, alkene lysine, and bipyridine lysine, were achieved through the pyrrolysyl-tRNA synthetase (PylRS). The UAA mutagenesis system was further studied, including optimization of the incorporation efficiency in mammalian cells, library selection of UAA in E. coli, and genetic incorporation of photocaged cysteine through E. coli Leu synthetase. UAA mutagenesis provides a versatile method for protein activity control and protein conjugation. As one application, optical control of protein activities (intein, TEV protease, Src kinase, HRP) was studied, with the genetic incorporation of photocaged or photoisomerizable UAAs. Optical deactivation of protein function was studied with degron or genetic incorporation of nitrophenylalanine. Besides, small molecule control of protein activity was studied with the genetic incorporation of a propargyl carbamate or an allyloxy carbonyl functional group. As another application, an aminooxy functional group was genetically incorporated for protein labeling. Next, the zebrafish with the expanded genetic code was developed, and was applied to the optical control of cell signaling (MEK/ERK pathway). Optical control of other proteins (Alk5 kinase, Cas9, Cre recombination) and small molecule control of luciferase function in zebrafish were studied. Efforts were also made to generate stable fishlines for genetic code expansion. Lastly, optical control of other biomolecules (morpholino, peptide nucleic acid) was pursued in zebrafish.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Liu, Jiheliujihe.2011@gmail.comjil147
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDeiters, Alexanderalexdeiters@gmail.com
Committee MemberHorne, Sethhorne@pitt.edu
Committee MemberChilders, Sethwschild@pitt.edu
Committee MemberTsang, Michaeltsang@pitt.edu
Date: 31 January 2018
Date Type: Publication
Defense Date: 17 July 2017
Approval Date: 31 January 2018
Submission Date: 20 November 2017
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 301
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: chemical biology, synthetic biology
Date Deposited: 31 Jan 2018 18:24
Last Modified: 31 Jan 2018 18:24
URI: http://d-scholarship.pitt.edu/id/eprint/33396

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