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Validation of interactions between aminoacyl- tRNA synthetases and cancer-associated factors using FRET-based screening system : FRET 기반의 screening system을 사용한 aminoacyl-tRNA synthetases와 cancer-associated factors의 상호작용 검증
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 김성훈 | - |
dc.contributor.author | 박보라진아 | - |
dc.date.accessioned | 2017-07-19T11:04:33Z | - |
dc.date.available | 2017-07-19T11:04:33Z | - |
dc.date.issued | 2013-02 | - |
dc.identifier.other | 000000010326 | - |
dc.identifier.uri | https://hdl.handle.net/10371/133331 | - |
dc.description | 학위논문 (석사)-- 서울대학교 융합과학기술대학원 : 분자의학 및 바이오제약학과, 2013. 2. 김성훈. | - |
dc.description.abstract | Aminoacyl-tRNA synthetases(ARSs) are essential proteins that are involved in cellular protein synthesis and viability. They catalyse the specific amino acids to their cognate tRNAs with a high fidelity. However, previous study shows that these enzymes are multi-functional proteins that are involved in diverse cellular processes. According to the recent study, a systematic analysis of the expression of ARSs indicates that these proteins are associated with cancer. Because of the need to identify the correlation, we developed a cell-based high throughput screen method using förster resonance energy transfer (FRET) for anticancer activity in the living cells. Fluorescence resonance energy transfer (FRET) is unique in generating fluorescence signals that are sensitive to molecular conformation, association, and separation in the 1-10nm range. At first, we constructed vectors that expressed cyan fluorescent protein (CFP) tagged ARSs and yellow fluorescent protein (YFP) tagged CAGs. Then we used a confocal microscope and calculated the FRET efficiency. As a result, we found that AIMP3 and RPSA protein pair which has higher FRET efficiency than the control. To confirm this protein-protein interaction, we conducted co-immunoprecipitation and in vitro pull down assay. From the previous experiments, we identified that the AIMP3 and RPSA has a direct interaction.
Accordingly, our results indicated that FRET-based validating system could be used to detect protein-protein interactions. Moreover, with our assay, it could help with the development of new anti-cancer drugs in the future. | - |
dc.description.tableofcontents | CONTENTS
ABSTRACT 1 CONTENTS 3 LIST OF FIGURES 5 LIST OF TABLES 6 I.INTRODUCTION 7 II.MATERIALS AND METHODS 10 1.Vector construction 10 2.Cell culture 11 3.DNA transfection 11 4.FRET assay 12 5.Co-immunoprecipitation and western blot analysis 12 6.GST fusion protein purification and in vitro pull-down assay 13 III. RESULT 15 1.Development of the optimized donor and acceptor pairs for FRET screening 15 2.Monitoring FRET signals in living mammalian cells 16 3.Validation of the FRET screening results 18 4.The improper orientation of AIMP3-CFP/RPSA-YFP indicates no FRET 19 5.The aspects of expression to consider for FRET occurrence 20 IV. DISCUSSION 32 V. REFERENCE 34 VI. 국문초록 37 | - |
dc.format | application/pdf | - |
dc.format.extent | 816494 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject.ddc | 610 | - |
dc.title | Validation of interactions between aminoacyl- tRNA synthetases and cancer-associated factors using FRET-based screening system | - |
dc.title.alternative | FRET 기반의 screening system을 사용한 aminoacyl-tRNA synthetases와 cancer-associated factors의 상호작용 검증 | - |
dc.type | Thesis | - |
dc.description.degree | Master | - |
dc.citation.pages | 38 | - |
dc.contributor.affiliation | 융합과학기술대학원 분자의학 및 바이오제약학과 | - |
dc.date.awarded | 2013-02 | - |
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