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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17977完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王彥士(Yane-Shih Wang) | |
| dc.contributor.author | Jia-Cheng Sun | en |
| dc.contributor.author | 孫嘉呈 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:47:34Z | - |
| dc.date.copyright | 2020-09-22 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17977 | - |
| dc.description.abstract | 基因密碼的擴充和轉譯重新定義在蛋白生合成中為擴展蛋白立體及化學多樣性的當代化學生物學及合生物學發展方向之一。蛋白生合成中以基因停止碼嵌入非典型胺基酸導入新式化學及物理性質，生物正交胺醯-核醣核酸合成酶‧轉核醣核酸(aaRS‧tRNA)配對在體內核醣體合成胜肽鏈，天然中可轉譯胺基酸被認為L-胺基酸，但活體外測試中，核醣體對於D-胺基酸芳香族側鏈受質在胜肽鍵合成中仍保有相對L-胺基酸50-70%的轉錄活性。過去本實驗室利用工程吡咯-轉核醣核酸合成酶(PylRS)及其tRNAPyl配對以大腸桿菌中大量生合成三種D-間位取代苯丙胺醯-tRNAPyl，成功活體內嵌入蛋白中。在此研究當中，探討演化樹鄰近另一群不具N端結構的∆PylRS，五種具備的生物正交配對∆PylRS‧tRNAPyl藉由互相配對相對正交性。測試結果顯示野生株Methanogenic archaeon ISO4-G1 (G1) PylRS‧Methanonatronarchaeum termitum (Mt) tRNAPyl異種配對可利用D-非典型胺基酸並和Methanosarcina mazei (Mm)的突變配對MmDFPylRS‧MmtRNAPyl相互正交，研究進一步以兩種不同的D-非典型胺基酸分別在超折疊綠螢光蛋白(sfGFP)中的指定的終止密碼子S2TAG和F27TAA位置嵌入相應胺基酸，在調控胺基酸濃度下，以質譜檢測12種D-非典型胺基酸組合蛋白轉譯正確性，同時在基因終止密碼子改變順序下仍保持正確轉譯。 | zh_TW |
| dc.description.abstract | Genetically incorporation of non-canonical amino acids (ncAAs) into proteins by expanding genetic code is a robust technique in synthetic biology to introduce new moieties to alter the chemical and physical properties of proteins. Numerous in vitro studies have proved that the native ribosomal pathway remains accepting D-aromatic amino acids, i. e. D-phenylalanine, D-tyrosine, and D-histidine, in 50 to 70% in peptide bond formation comparing their L-isomers. Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS) ‧tRNAPyl pair was evoloved to incorporate multiple meta-substituted D-phenalanines in Escherichia coli. In this study, a novel clade of ∆PylRS‧tRNAPyl pairs, which was functioned without N-terminal domain. These five ∆PylRS‧tRNAPyl pairs were reported great bioorthogonality in E. coli. Among these enzymes and combinations, wild-type Methanogenic archaeon ISO4-G1 (G1) PylRS‧Methanonatronarchaeum termitum (Mt) tRNAPyl heterogentic pair displays several D-ncAA compatibility in substrate range screening. G1PylRS‧MttRNAPyl pair was utilized in companion with previous evolved Methanosarcina mazei (Mm) PylRS‧MmtRNAPyl pair (MmDFPylRS‧MmtRNAPyl) to establish mutually orthogonal pairs. Two distinct DncAAs were successfully incorporated onto the designated positions of superfolder green fluorescence protein (sfGFP) with S2TAG/F27TAA or S2TAA/F27TAG stop codon assignments. Codon transaltion fidelity and efficiency at stop codons, TAG and TAA, were investigated in sfGFP production and confirmed by full-length protein mass spectrometry by these two PylRS‧tRNAPyl pairs. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:47:34Z (GMT). No. of bitstreams: 1 U0001-1308202018455700.pdf: 11932281 bytes, checksum: e2b1628e7b0b1300ddaf1eed5b38f548 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 摘要 I Abstract II Table of contents III List of figures V List of tables VII List of scheme VIII Abbreviations IX Chapter 1 Introduction 1 1.1 Protein translation machinery in prokaryotes 1 1.2 Expanding genetic code 2 1.3 D-amino acids incorporation 5 1.4 Multiple and different noncanonical amino acid incorporation and applications 9 1.5 Specific aims 12 Chapter 2 Materials and methods 13 2.1 plasmid construction 13 2.2 Substrate range investigation by screening 16 2.3 Protein expression and purification 17 2.4 Gel analysis 18 2.4.1 SDS-PAGE analysis 18 2.4.2 Western blot analysis 19 2.5 Protein ESI-MS analysis 20 Chapter 3 Results 21 3.1 Phylogenetic analysis of PylRS and tRNA 21 3.2 Two DncAAs incorporation 22 3.2.1 Establishment of mutually orthogonal PylRS‧tRNAPyl pairs 22 3.2.2 Codon fidelity and translation efficiency of selected pairs 25 3.2.3 ESI-MS analysis of sfGFP variants 27 Chapter 4 Discussion 30 Chapter 5 Conclusion 32 Reference 65 Table of contents for appendix 71 Appendix 74 | |
| dc.language.iso | en | |
| dc.title | 相異兩種D-非典型胺基酸嵌入蛋白生合成之研究 | zh_TW |
| dc.title | Genetic incorporation of two distinct D-noncanonical amino acids | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.advisor-orcid | 王彥士(0000-0003-2356-9577) | |
| dc.contributor.oralexamcommittee | 蔡明道(Ming-Daw Tsai),王健家(Chien-Chia Wang) | |
| dc.contributor.oralexamcommittee-orcid | 蔡明道(0000-0003-1374-0414),王健家(0000-0002-7104-1307) | |
| dc.subject.keyword | D-非典型胺基酸,吡咯-轉核醣核酸合成酶,相互生物正交配對, | zh_TW |
| dc.subject.keyword | D-noncanonical amino acids,Pyrrolysyl-tRNA synthetase,Mutually orthogonal PylRS‧tRNAPyl pairs, | en |
| dc.relation.page | 203 | |
| dc.identifier.doi | 10.6342/NTU202003313 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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