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Cell type-specific proteome labeling by genetic code expansion

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dc.contributor.author김은진-
dc.date.accessioned2017-07-07T16:10:44Z-
dc.date.available2017-07-07T16:10:44Z-
dc.date.issued2016-
dc.identifier.urihttps://ir.ymlib.yonsei.ac.kr/handle/22282913/148838-
dc.description의과대학/석사-
dc.description.abstractAnalyzing gene expression of specific cells in a living organism is crucial in understanding complex biological systems. It has not been possible, however, to isolate the proteome of specific cells in vertebrates. Recently, advance in genetic code expansion has provided a new potential direction in achieving this aim. By using the tool of genetic code expansion, I expanded the genetic code of mammalian cells by introducing the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl system of Methanosarcina mazei, a methane-producing genus of archaea. Although the amber (UAG) codon is a stop codon to most of organisms, Methanosarcina uses it as a sense codon that encodes pyrrolysine (Pyl), the 22nd amino acid. Unlike eukaryotes where the amber codon is recognized by eukaryotic release factor 1 (eRF1) which helps the termination of mRNA translation, Methanosarcina expresses the tRNA (tRNAPyl) whose anticodon recognizes the amber codon. PylRS esterifies tRNAPyl and Pyl, generating the Pyl-tRNAPyl that site-specifically incorporates Pyl into the growing polypeptides during mRNA translation. In this thesis, I expressed PylRS and tRNAPyl in mammalian cells and showed that the M. mazei PylRS/tRNAPyl system is functional in mammalian cells and orthogonal to endogenous tRNAs and tRNA synthetases. I also showed that the anticodon of tRNAPyl can be changed to recognize other stop and sense codons without compromising their compatibility with PylRS and Pyl. Furthermore, I showed that Pyl-derivatives containing bio-orthogonal functional group such as alkyne can be site-specifically incorporated into the endogenous proteome only when the genetic code was expanded by the PylRS/tRNAPyl system. By using azide-alkyne cycloaddition, the de novo proteome of genetic code-expanded cells could be specifically visualized. The results of this thesis show a promising direction for a new technique to isolate the proteome of a specific cell type from a living animal.-
dc.description.statementOfResponsibilityopen-
dc.publisherGraduate School, Yonsei University-
dc.rightsCC BY-NC-ND 2.0 KR-
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/2.0/kr/-
dc.titleCell type-specific proteome labeling by genetic code expansion-
dc.typeThesis-
dc.contributor.alternativeNameKim, Eunjin-
dc.type.localThesis-
Appears in Collections:
1. College of Medicine (의과대학) > Others (기타) > 2. Thesis

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