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研究生:盧佑成
論文名稱:比較自然型式與人造分離式內含蛋白對洗滌劑及變性劑之抗性
論文名稱(外文):Comparison of Natural and Artificial Split Intein in Resisting Detergents and Denaturants
指導教授:蘇士哲蘇士哲引用關係
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:54
中文關鍵詞:內含蛋白蛋白質反式剪接多肽固相合 成法氫鍵電荷交互關係
外文關鍵詞:InteinProtein trans-splicingSolid phase peptide synthesisHydrogen bondCharge-charge interaction
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蛋白質剪接
(Protein
splicing)
作用是一種經由內含蛋白(intein)能夠自我催
化進行的蛋白質連接過程,蛋白質剪接能夠連接N端以及C端的外顯蛋白(extein)
序列並且切除內含蛋白(intein)本身。這個過程有兩種方式,剪接作用若發生在單
一條多胜肽片段上稱之為順式剪接作用(cis-splicing)
,相較下反式剪接作用
(trans-splicing)則是利用分離式內含蛋白,且使得兩部分蛋白質片段組合起來。
此外蛋白質反式剪接作用不僅在活體內被發現,也能夠在細胞體外進行。至今,
蛋白質反式剪接作用在生物科技應用上像是特定點位標訂的生物物理探針,核磁
共振部分同位素標訂法於蛋白質結構或動力學研究,蛋白質半合成及其後轉譯修
飾,以及蛋白質環化作用。蛋白質反式剪接作用亦闡述透過核磁共振,利用部分
同位素標定法來決定其蛋白質結構為基礎的可能性。為了使得各項基於蛋白質反
式剪接作用的生物應用能活用在各種形式的蛋白質上,因此蛋白質反式剪接作用
能否行使在各種條件環境下就顯得額外重要。順帶一題,先前的研究曾表述自然
形式的Nostoc
punctiforme
(Npu)
dnaE 分離式蛋白質內含子(Npu102)在洗滌劑
或是變性劑的存在下擁有絕佳的耐受性。在本次研究中,我們試驗著新式開發設
計的Nostoc
punctiforme
(Npu)
dnaE 分離式蛋白質內含子(Npu36)在尿素、胍
鹽酸以及各式洗滌劑進行蛋白質反式剪接作用能力。新式開發設計的分離式蛋白
質內含子(Npu36)是由自然形式的分離式蛋白質內含子(Npu102)演變設計而
來。相較於自然形式的分離式蛋白質內含子(Npu102),新式開發設計的分離式
蛋白質內含子(Npu36)擁有著較短的N-片段。這使得新式開發設計的分離式蛋
白質內含子(Npu36)的N-片段在多肽固相合成法擁有較佳的潛力,原因來自於
較短的片段能夠有力的應用在多肽固相合成法中。此外,我們也利用所有的資料
證明N和C片段中氫鍵以及電荷交互關係比起整體的結構更具影響蛋白質反式
剪接作用。
The protein splicing is an autocatalytic protein ligation process by intein. The protein splicing ligates the N- and C-terminal flanking sequence, termed “extein”, and excised intein domain itself. The process comes in two flavors. Splicing reaction in single polypeptide chain from one component in terms of cis-splicing. By contrast, the trans-splicing associated two components by using split inteins. Furthermore, the PTS not only occur in vivo but also enable in vitro. Until recently, the biological application of protein trans-splicing (PTS) includes site-specific incorporation of biophysical probes, segmental isotope labeling for NMR structure study or dynamics research, protein semi-synthesis containing posttranslational modifications, and protein cyclization. PTS also elicits a possibility from segmental isotope labeling and macromolecular protein structure determination by solution NMR. To precede the PTS reactions under different environments becomes a concerned issue for split intein. The previous data indicated that nature Nostoc punctiforme (Npu) DnaE split intein (Npu102) has great tolerance in the presence of either detergents or denaturants. In our study, we examined trans-splicing activity of a newly engineering Npu DnaE split intein (Npu36) in the presence of urea, guanidine hydrochloride or detergents. In comparing to Npu102, the N-terminal intein fragment of Npu36 was shorter than that in Npu102. Therefore, the short N-terminal portion provides benefit in PTS if synthesis of N-terminal intein fragment is required. In the study, we demonstrate the hydrogen bonds and charge-charge interaction between N- and C-terminal intein fragments of split intein make significant contributions than overall structure of split intein for PTS.
謝誌.......................................................3
中文摘要....................................................4
Abstract...................................................5
Introduction...............................................6
- NMR strategy : Segmental isotope labeling................6
- Intein and split intein..................................8
- The chemical mechanism of protein-trans splicing........11
- Solid Phase Peptide Synthesis...........................12
- The new split site at native Npu DnaE split intein......15
Materials and methods.....................................18
- Cloning and construction-Npu102, Npu36, CPSP102 & CPSP36....................................................18
- Protein expression and purification.....................23
- Split intein for protein trans-splicing assay in vitro..24
- Kinetic analysis of protein trans-splicing in the presence of denaturants............................................25
- Kinetic analysis of protein trans-splicing in the presence of detergents.............................................26
- To prepare and analyze the titration experiment and backbone measurement by NMR...............................28
- To prepare and analyze the structure of Npu36...........28
Results and discussions...................................29
- Splicing kinetics in the presence of Urea and GdmCl comparison of native and artificial Npu DnaE split intein.29
- NMR HSQC of Npu36 and Npu102 in the presence of urea reveals the structure information correlate to the PTS ability...................................................30
- Splicing kinetics in the presence of different detergents comparison of Npu102 and Npu36 split intein...............31
- Analysis and comparison between the Npu102 and Npu36 split intein....................................................32
Conclusion................................................34
References................................................53
1. Muona, M., Aranko, A. S., Raulinaitis, V. & Iwai, H. (2010). Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro. Nat Protoc 5, 574-87.
2. Chen, J. & Wang, J. (2011). A segmental labeling strategy for unambiguous determination of domain-domain interactions of large multi-domain proteins. J Biomol NMR 50, 403-10.
3. Skrisovska, L., Schubert, M. & Allain, F. H. (2010). Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins. J Biomol NMR 46, 51-65.
4. Shah, N. H. & Muir, T. W. (2011). Split Inteins: Nature's Protein Ligases. Isr J Chem 51, 854-861.
5. Volkmann, G. & Mootz, H. D. (2013). Recent progress in intein research: from mechanism to directed evolution and applications. Cell Mol Life Sci 70, 1185-206.
6. Anraku, Y., Mizutani, R. & Satow, Y. (2005). Protein splicing: its discovery and structural insight into novel chemical mechanisms. IUBMB Life 57, 563-74.
7. Saleh, L. & Perler, F. B. (2006). Protein splicing in cis and in trans. Chem Rec 6, 183-93.
8. Muir, T. W., Sondhi, D. & Cole, P. A. (1998). Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci U S A 95, 6705-10.
9. Severinov, K. & Muir, T. W. (1998). Expressed protein ligation, a novel method for studying protein-protein interactions in transcription. J Biol Chem 273, 16205-9.
10. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. (1994). Synthesis of proteins by native chemical ligation. Science 266, 776-9.
11. Evans, T. C., Jr., Benner, J. & Xu, M. Q. (1998). Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci 7, 2256-64.
12. Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E., Neff, N., Noren, C. J., Thorner, J. & Belfort, M. (1994). Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature. Nucleic Acids Res 22, 1125-7.
13. Clarke, N. D. (1994). A proposed mechanism for the self-splicing of proteins. Proc Natl Acad Sci U S A 91, 11084-8.
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15. Xu, M. Q. & Perler, F. B. (1996). The mechanism of protein splicing and its modulation by mutation. EMBO J 15, 5146-53.
16. Merrifield, R. B. (1963). Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Journal of the American Chemical Society 85, 2149-2154.
17. Sun, W., Yang, J. & Liu, X. Q. (2004). Synthetic two-piece and three-piece split inteins for protein trans-splicing. J Biol Chem 279, 35281-6.
18. Vila-Perello, M. & Muir, T. W. (2010). Biological applications of protein splicing. Cell 143, 191-200.
19. Lee, Y. T., Su, T. H., Lo, W. C., Lyu, P. C. & Sue, S. C. (2012). Circular permutation prediction reveals a viable backbone disconnection for split proteins: an approach in identifying a new functional split intein. PLoS One 7, e43820.
20. Lo, W. C., Dai, T., Liu, Y. Y., Wang, L. F., Hwang, J. K. & Lyu, P. C. (2012). Deciphering the preference and predicting the viability of circular permutations in proteins. PLoS One 7, e31791.
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23. Wu, H., Hu, Z. & Liu, X. Q. (1998). Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A 95, 9226-31.

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