臺灣博碩士論文加值系統

第一型綠豆防禦素(VrD1)與第二型綠豆防禦素(VrD2)被歸類成植物防禦素。植物防禦素是一種小分子胜肽，會形成四對雙硫鍵來穩定結構。主要的作用是做為植物免疫的第一道防線，三級結構與其他生物體內的防禦素非常相似，對於植物及動物細胞不會產生毒性。先前的研究將VrD1第三個loop的序列置換到VrD2的第三個loop上，創造了一個新的嵌合蛋白質VrD2c能帶有抑制昆蟲澱粉酶的功能。基於這樣的研究成果，我們希望利用蛋白質工程的技術，給予VrD2c第二項功能。
在本次的研究，將胰蛋白抑制劑的序列轉到VrD2c的Loop 1上做為第二項功能，同時也計畫將RGD序列轉移到VrD2的loop1及loop 2上，期望藉由與integrin高度結合，成為治療血小板疾病的蛋白質藥物。我們利用圓二色光譜儀觀察到變異蛋白均展現高度的穩定性，新的嵌合蛋白同時帶有抑制胰蛋白酶及澱粉酶的功能。此外，我們計畫利用血小板聚集試驗，確認帶有RGD序列的VrD2衍生物具抑制血小板凝集的功能。就目前結果來看，VrD2是一個適合用來進行蛋白質工程的支架蛋白，我們可以將VrD2的loop置換成不同的序列，以期能開發為帶有特殊性質的新蛋白。

Abstract 1
中文摘要 2
Abbreviations 3
Chapter 1. Introduction 4
Part I: Trypsin Inhibitor design 4
1.1 Plant Defensin 4
1.2 Vigna Radiata Plant Defensin 2 5
1.3 Trypsin Inhibitor 5
1.4 Specific aims 7
Part II: Anti-Clotting Protein Design 8
1.1 Integrin αIIbβ3 8
1.2 RGD Sequence 8
1.3 The motivation of this part 9
Chapter2. Materials and Methods 10
2.1 Construction of VrD2 mutants 10
2.2 Protein expression and purification 10
2.3 Purification and activity assay of Tenebrio molitor α-amylase 11
2.4 Tricine SDS-PAGE 12
2.5 MASS analysis 13
2.6 Quantification of protein concentration 14
2.7 Circular Dichroism (CD) Experiments 14
2.8 Trypsin inhibition Assay 15
2.9 Tenebrio molitor α-amylase Inhibition Assay 15
2.10 Molecular Modeling and Docking. 16
Chapter 3. Results and Discussion 17
Part I: Trypsin Inhibitor design 17
3.1 Protein Design, Molecular Modeling and Docking 17
3.2 Protein expressions and purification of VrD2c-1Y 17
3.3 Secondary Structure comparison of VrD2c-1Y and VrD2 18
3.4 Trypsin Inhibitory assay 19
3.5 Purification of TMA and α-Amylase Inhibitory Assay 19
Part II: Anti-Clotting Protein Design 21
3.1 Target Selection for RGD Design 21
3.2 Protein expressions and purification of D2R1 and D2R2 21
3.3 Secondary Structure of RGD proteins 21
Chapter 4. Conclusion 23
Tables and Figures 25
Figure 3.1 Strategy of trypsin inhibitor design 25
Figure 3.2 Molecular model and Ramachandran Plot analysis of VrD2c-1Y 26
Figure 3.3 Molecular docking model 27
Figure 3.4 VrD2C-1Y expression was analyzed by SDS-PAGE 28
Figure 3.5 VrD2C-1Y purification analysis by SDS-PAGE 29
Figure 3.6 HPLC profiles of VrD2c-1Y 30
Figure 3.7 Mass spectra determination of VrD2c-1Y 31
Figure 3.8 Far-UV CD spectra of VrD2c-1Y, VrD2 and VrD2c. 32
Figure 3.9 Far-UV CD spectra of VrD2c-1Y, trypsin and 1Y-trypsin complex. 33
Figure 3.10 Far-UV CD spectra of VrD2c-1Y in PBS buffer with and without TFE. 34
Figure 3.11 Inhibitory activity of VrD2c-1Y against Trypsin 35
Figure 3.12 Purification of TMA on FPLC system 36
Figure 3.13 TMA purification analysis by SDS-PAGE. 37
Figure 3.14 Inhibitory activity of VrD2c-1Y against TMA 38
Figure 3.15 Strategy of protein design containing RGD sequence 39
Figure 3.16 RGD proteins expression was analyzed by SDS-PAGE 40
Figure 3.17 RGD proteins purification analysis by SDS-PAGE. 41
Figure 3.18 HPLC profiles of RGD protein. 42
Figure 3.20 Far-UV CD spectra of D2R1, D2R2 and VrD2. 44
Figure 3.21 Far-UV CD spectra of D2R2 in PBS buffer with and without TFE. 45
Table 1. The properties of VrD protein which we used in this study 46
Table 2. Sequence of oligonucleotides used for site-directed mutagenesis. 46
Appendix 1 47
Appendix 2 48
Appendix 3 49
Reference 50

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