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研究生： 蘇子翔
論文名稱： 分離式內含蛋白以協助研究膜蛋白結構
Split intein in support of membrane protein structural study
指導教授： 蘇士哲
口試委員: 陳金榜
黃太煌
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2012
畢業學年度： 100
語文別： 英文
論文頁數： 62
中文關鍵詞： 內含蛋白 、蛋白質反式剪接 、部分同位素標定法 、外膜蛋白
外文關鍵詞： intein, protein trans splicing, segmental isotope labeling, outer-membrane protein X
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    • 蛋白質剪接 (Protein splicing) 作用是一種自我催化的過程，它能連接兩端的外顯蛋白(extein)序列而釋放內含蛋白(intein)本身。這個過程有兩種方式，剪接作用發生在單一條多胜肽片段上則稱為順式剪接作用(cis-splicing) ，相較於反式剪接作用則是利用分離式內含蛋白，將兩部分蛋白質片段組合起來。蛋白質反式剪接作用在生物科技運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。以Nostoc punctiforme (Npu) dnaE分離式蛋白質內含子支架為基礎，透過分離式蛋白質內含子的剪接作用功能，我們成功的表現目標膜蛋白，藉由兩已分離的互補片段並在試管中將之連接在一起。除此之外，我們也注意到Npu分離式蛋白質內含子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，我們將完整的膜蛋白Outer membrane protein X(OmpX) 拆開成兩個片段，且於4M尿素的環境下成功的接合。已接合的蛋白質產物在DHPC洗滌劑環境下重新摺疊，並透過膠體移動試驗來檢查是否有重新摺疊。相關的核磁共振實驗仍在持續進行中，我們相信這樣的策略在將來能夠運用到大型膜蛋白上。

    Protein splicing is an autocatalytic molecular process that ligates the N- and C-terminal flanking sequence, called “extein”, and liberates “intein” domain itself. This process comes in two flavors. Splicing reaction in single polypeptide from one component in terms of cis-splicing compared trans-splicing that associated two components by using split inteins. The biological application of protein trans-splicing (PTS) such as site-specific incorporation of biophysical probes, segmental isotope labeling for NMR structure or dynamics research, protein semi-synthesis containing posttranslational modifications, and protein cyclization. Protein trans-splicing also elicits a possibility from segmental isotope labeling and macromolecular protein structure determination by solution NMR, base on the Nostoc punctiforme (Npu) dnaE split intein scaffold, we successfully express the target membrane protein by two separated complementary pieces and ligate the fragments in vitro based on the trans-splicing function of split intein. Besides, we also noticed that Npu split intein has great tolerance in the presence of either detergents or denaturants. In this study, we divided full-length outer-membrane protein X (OmpX) into two fragments and successfully ligated in 4 Molar urea conditions. The ligated product refolded in DHPC detergent and checked by gel shift assay. The related NMR experiment is currently ongoing. We believe this strategy can be applied to large membrane proteins in near future.

    謝誌 3 中文摘要 5 Abstract 6 Introduction 7 - NMR strategy : Segmental isotope labeling……………………………………….………7 - Intein and split intein: The versatile tools for protein ligation……........…...….………...9 - General organization of an intein……………………………………………….………….10 - Current views of major chemical mechanism of protein splicing…………...……….....12 - Structural studies of membrane proteins by NMR methods..........................…..........15 - Protein trans-splicing and its biotechnologies applications……………….....…...........16 Materials and methods 18 - Expression and purification of native split intein and artificial split intein……............18 - Split intein for protein trans-splicing assay in vitro…………………………………......21 - Protein trans-splicing assay in presence of detergents and denaturants………........23 - Point mutation of split intein for isothermal titration calorimetry………….…..............24 - CPred: Circular Permutation site predictor………………………………………..…….27 - Expression and purification of OmpX and split intein fused OmpX………..…….…...28 - Preparation and analysis for solution NMR spectroscopy….…………….…….…......31 Results and discussions 32 - Splicing kinetics in trans using naturally split intein with novel splitting sites…….....32 - Binding affinity comparison of two split inteins by ITC experiments…….…………....33 - Splicing kinetics in the presence of denaturants and detergents………..…..…….....34 - NMR spectrum reveals the structural information in the presence of urea……….....35 - Naturally split intein in support of split OmpX ligation in vitro………………..…….....36 Conclusion 37 References 57 Appendix 61

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