白血球介素-2（Interleukin-2, IL-2）是一種15.5 kDa的細胞因子，由四個α螺旋所組成。IL-2會影響不同淋巴細胞的增生或是分化進而控制與維持體內的免疫反應。因此IL-2已被認為是有效的免疫細胞治療藥物。IL-2在臨床治療上，因為其半衰期短以及在施打的劑量過多易產生細胞毒性。為了克服這些限制，我們設計了能夠提升結構穩定性的IL-2突變體。在我們的研究中，首先使用電腦軟體來預測可能增強 IL-2 穩定性的單點突變體。我們以IL-2晶體結構為軟體的預測模板，並刪去可能會影響到與受體結合的突變位點後，最後我們選擇了三個單點突變體，分別是P82W、Q74G和K97W。將野生型的IL-2與其突變體蛋白經由純化並確認其分子量後，再透過圓二色光譜儀 (circular dichroism, CD) 分析其二級結構。在CD實驗中我們分別測試了他們在不同pH值 條件下的全波長 (195-260 nm)光譜，並透過解構溫度 (melting temperature, Tm)比較IL-2蛋白熱穩定性。從圓二色光譜儀的實驗結果得知，與野生型IL-2相比，經電腦預測後所選的單點突變體具有增強α螺旋的特性，尤其突變體P82W擁有最明顯的α螺旋特性。除此之外，我們還發現了賦形劑具有增加IL-2溶解度的能力。添加賦形劑後可能能夠阻止IL-2在高濃度下產生的聚集反應並進一步增強了蛋白質穩定性。最後再藉由核磁共振儀 (nuclear magnetic resonance, NMR)了解IL-2野生型與突變體P82W在不同pH值條件下對蛋白質三級結構的影響，我們發現蛋白在酸性環境下容易產生聚集，但將pH值提升到pH 5，蛋白擁有最佳的折疊情形，且P82W突變體的光譜比野生型更佳，代表其穩定性更好，具有潛力作為未來在臨床治療上的藥物。

Interleukin-2 (IL-2) is a 15.5 kDa cytokine consisting of a structure of four-helix bundle. Different lymphocyte subsets are influenced by IL-2 during differentiation, immunological responses, and homeostasis. Thus, IL-2 has been developed as an effective drug used in immune cell therapy. However, IL-2's clinical utility is limited by its short half-life and concentration-dependent pleiotropic effects. To overcome the limitation, we consider designing an “upgraded” IL-2 mutant with better structural stability. Using the IL-2 crystal structure as the template and filtering out the mutants that might influence the receptor binding, we selected three single-site mutations, which are P82W, Q74G, and K97W, possibly bringing the negative free energy changes (∆G < 0) for structure. We constructed and expressed the IL-2 mutants and tested their thermal stability by circular dichroism (CD) spectrum. We acquired the full wavelength (195-260 nm) in different pH conditions and determined the melting temperature (Tm). The CD study demonstrated the selected mutants with enhanced helical properties compared to the native IL-2, especially for the mutant P82W with the highest helical property. Additionally, we identified an excipient to increase IL-2 solubility. The additive might prevent IL-2 aggregation in high concentrations and further enhance protein stability. In final, we used NMR to study the pH effects of WT and P82W and their tertiary structures. We found that WT and P82W aggregate in an acidic environment. When the pH value is increased to 5, the protein has a well folding state and the P82W has a better spectrum than the WT. The predicted mutation, P82W, may be useful in designing a more stable IL-2 for future clinical usage.

Abstract II
中文摘要 III
Acknowledgement IV
Abbreviations VI
Chapter 1. Introduction 1
1.1 Interleukin 1
1.1.1 Interleukin-2 1
1.2 The interleukin-2 receptor 2
1.3 The current therapy problem of Interleukin-2 3
1.4 The aim of the study 4
Chapter 2. Materials and methods 9
2.1 Stable mutant structure predict by Rosetta program 9
2.2 Cloning and expression of Interleukin-2 and mutants 9
2.3 Purification of Interleukin-2 and mutants 9
2.4 Isotope label protein culture 10
2.5 Tobacco Etch Virus (TEV) protease purification 10
2.6 Circular dichroism (CD) 11
2.7 pH titration experiment of NMR 11
Chapter 3. Results 13
3.1 Predicted result of Rosetta program 13
3.2 Interleukin-2 and mutant purification 14
3.3 CD experiment of IL-2 and mutants 15
3.3.1 CD buffer condition 15
3.3.2 CD profiles of WT and mutants 15
3.3.3 Tm experiment 16
3.3.4 The structural stable IL-2 P82W 17
3.4 NMR 1H-15N HSQC of WT and P82W 18
Chapter 4. Discussion 41
References 46
Appendix 48

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