環形重組是一種改善蛋白質特性的蛋白質工程技術。和傳統的突變相比，環形重組是以連接胜肽 (linker) 聯繫原本的胺基端和羧基端，再打開新的端點，造成蛋白質序列排列改變。綠豆植物防禦素第一型 (VrD1) 是個富含半胱胺酸的蛋白，共有四對雙硫鍵，其中有對雙硫鍵 (C3-C46) 連結了原本的胺基端和羧基端。我們將環形序列重組的蛋白質工程技術應用到綠豆植物防禦素第一型，去探討連接胜肽的長度對富含雙硫鍵的蛋白質之影響。透過圓二光偏極光譜儀，內生性螢光偵測，及抑制澱粉酶活性測定來分析環形重組綠豆植物防禦素第一型的結構與功能變化。我們發現連接胜肽短於原本胺基端和羧基端的距離會造成蛋白質結構改變且喪失抑制澱粉酶的功能。因此我們推測在使用較短連接胜肽的環形重組綠豆植物防禦素第一型上所觀察到的蛋白質結構變化可能是因為較短的連接胜肽及終端的雙硫鍵 (C3-C46) 同時存在造成局部較高的約束力，因而導致蛋白質折疊不佳。我們利用定點突變的方法，進一步將不同長度的連接胜肽接合的環形重組綠豆植物防禦素第一型中的終端的雙硫鍵 (C3-C46) 移除。結果顯示移除終端的雙硫鍵 (C3-C46) 的環形重組綠豆植物防禦素第一型其蛋白質摺疊仍然不佳，且熱穩定度下降，但不會影響其功能。因此我們認為對於環形重組綠豆植物防禦素第一型，連接胜肽的長度所造成的約束力，比起終端雙硫鍵 (C3-C46) 來得重要。

Circular permutation (CP) is a protein-engineering technique to improve the characteristics of protein. In contrast with traditional mutagenesis, CP requires a linker to connect the native termini and creates new termini to result in the rearrangement of protein sequence. Vigna radiata plant defensin 1 (VrD1) is a cysteine-rich protein containing four disulfide bonds, and has a disulfide bond (C3-C46) bridging the native termini. We applied CP to VrD1 for exploring the influence of linker length and the effect of CP on cysteine-rich protein. The characteristics of structure and function in CP-VrD1 were analyzed through circular dichroism, intrinsic fluorescence, and α-amylase inhibition assay. We found that linkers with length less than the distance between original termini resulted in structural change and losing functions. Therefore, we speculated the poor folding of CP-VrD1 with shorter linker was due to the simultaneous existence of shorter linker and the terminal disulfide bond (C3-C46). Site-directed mutagenesis was used to remove terminal disulfide-bond in CP-VrD1 with linkers of different length, and to further elucidate the influence of linker length and disulfide-bond. Our results showed that terminal disulfide-bond removed CP-VrD1 still has poor structural folding and less thermal stability, but the function was not changed. We concluded that poor folding of CP-VrD1 was caused by the constraint of linker with shorter length rather than the existence of terminal disulfide bond (C3-C46).

Abbreviations 1
Chapter I Introduction 2
1.1 Plant Defensins 2
1.2 Vigna radiata plant defensin 1 (VrD1) 3
1.3 Circular permutation (CP) 4
1.4 The theme of the thesis 6
Chapter II Materials and Methods 8
2.1 Construction of circular-permuted VrD1 (CP-VrD1) 8
2.1.1 Materials 8
2.1.2 Methods 8
2.2 Expression and purification of CP-VrD1 9
2.2.1 Materials 9
2.2.2 Methods 9
2.3 Purification of Tenebrio molitorα-amylase (TMA) 11
2.3.1 Materials 11
2.3.2 Methods 11
2.4 Tricine-SDS-PAGE 12
2.5 Mass spectrometer 13
2.6 Quantification of protein concentration 13
2.7 Circular dichroism (CD) spectroscopy 14
2.8 Fluorescence spectroscopy 14
2.9 TMA activity assay and inhibition assay 15
Chapter III Results and Discussion 17
3.1 Design and Contruction of CP36-VrD1 17
3.2 Expression and purification of CP36-VrD1 18
3.3 Structural analysis of WT-VrD1 and CP36-VrD1 18
3.4 Thermal stability of WT-VrD1 and CP-VrD1 20
3.5 TMA-inhibition assay of VrD1 proteins 21
Chapter IV Conclusion 24
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