分離式內含蛋白 (split intein) 被廣泛的應用於蛋白質工程上，其中念珠藻(Nostoc punctiforme, Npu)內所發現的內含蛋白具有較好的反式剪接蛋白質接合(protein trans-splicing)活性。同時，此內含蛋白是少數被發現自然發生的斷口(naturally occurring split site). 此篇論文，將以 Npu DnaE intein 為研究目標，研究Npu DnaE split intein 之結構特性與 protein trans-splicing活性特性。首先的研究內容是利用環狀重排預測系統去尋找其他人工的斷口(artificial split site)，此預測方式未來可以運用於找尋其他新發現內含蛋白的斷口。接下來我們利用環狀重排技術制備Npu DnaE split intein 並利用核磁共振與X-ray來探討蛋白質的液態與晶體結構。再探討完結構分析後，我們利用Npu DnaE split intein於不同的洗滌劑與變性劑下進行反式剪接蛋白質接合活性測試。結果發現於多數的洗滌劑與變性劑下，Npu DnaE split intein仍然保有反式剪接蛋白質接合活性，除了帶電的洗滌劑與變性劑。同時我們在結構裡面發現，Npu DnaE split intein在結合的表面上有形成六組鹽橋(salt bridge)。當Npu DnaE split intein於高鹽環境結合時，此六組鹽橋會受到鹽的影響造成結合力下降與活性下降。最後我們利用Npu DnaE intein對鹽的靈敏度進而調控C端的分離反應。

Split inteins have been widely applied in protein engineering, and the Nostoc punctiforme (Npu) DnaE split intein has been reported to have outstanding activity. To expand usage of split intein, we firstly used a circular permutation predictor (CPred) to assist discovery of new artificial split inteins. Furthermore, we customized a method based on circular permutation (CP) concept to efficiently prepare naturally occurred split intein of NpuDnaE (NpuInt102) for NMR and X-Ray study. Crystal structure of NpuInt102 was solved and represented a structural state after protein trans-splicing. The structure has high similarity to one-fragment intein. However, few structural differences occurred in the opening split site and catalytic core. To evaluate the potential in applying split intein to membrane proteins, we tested protein trans-splicing under hydrophobic environments and found that intein could well perform protein trans-splicing, except in the presence of ionic solutes. The crystal structure of NpuInt102 indicated that 6 salt-bridges distribute on the assembling interface. The ionic solutes might impede the assembling between the two fragments by reducing electrostatic force. Based on the sensitivity to ions, we employed this feature to make intein to be a great fusion tag that the cleavage could easily remove from target proteins, regulated by salt concentration.

ABSTRACT I
摘要 II
ACKNOWLEDGMENT III
ABBREVIATIONS IV
Chapter I: General introduction of protein splicing 1
Chapter II: Applying circular permutation to identify the new artificial NpuDnaE split intein 11
II.1 Circular permutation 11
II.2 Circular Permutation Prediction (CPred) 11
II.3 Characterization of residues at CP sites 12
II.4 Using CPred to identify the new artificial NpuDnaE split intein 14
II.5 PRECEDURE 15
II.6 Case study 17
II.7 Summary 18
Chapter III: Protein preparation and structure of NpuDnaE and NpuDnaE102 24
III.1 The traditional method for preparation of split intein 24
III.2 A streamline method for preparation of split intein 27
III.3 Structure study in solution NMR 29
III.4 Solution structure comparison of NpuDnaE and NpuDnaE102 30
III.5 Crystal structure of NpuDnaE102 32
III.6 Conclusion 33
Chapter IV: Structural basis of detergent- and denaturant-resistant split intein 44
IV.1 Protein trans-splicing of naturally and artificial split inteins 45
IV.2 Splicing kinetics of split inteins in the presence of denaturants 46
IV.3 Trans-splicing kinetics in the presence of detergents 47
IV.4 Salt-bridges on interface 48
IV.5 Ionic strength affecting protein ligation activity 49
IV.6 Structure comparison between NpuInt102 and HutMCM2Intsplit 50
IV.7 Applying protein trans-splicing to ligate OmpX under denaturant 51
IV.8 Potential in applying on membrane proteins 51
IV.9 Conclusion 52
Chapter V: The application of side reactions during protein splicing 65
V.1 Cleavage of protein splicing 65
V.2 Tag-less protein splicing 66
V.3 Discussion 67
Chapter VI: Addition information for experimental details 74
VI.1 The concept of CPred program 74
VI.2 Protein expression and purification 75
VI.3 Protein trans-splicing assay in vitro 76
VI.4 Isothermal titration calorimetry (ITC) 78
VI.5 NMR measurement and resonance assignment. 78
VI.6 NMR 1H-15N HSQC spectrum and hydrogen/deuterium exchange experiment 79
VI.7 Crystallization, data collection and processing 80
VI.8 OmpX ligation 81

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