內含肽(Intein)具有連接肽骨架的能力，這樣的作用機制能夠延伸運用在環化多肽和蛋白質。根據過去的文獻提及環化的蛋白質具有較優異的抗熱性且在水溶液中表現得非常穩定，因此內含肽介導的分子內反應成為蛋白質工程中的常用且熱門的策略。在本篇的研究中，我們欲透過內含肽環化策略來形成環狀的第II型熱不穩定腸毒素B次單元，簡稱LTIIb-B5。為了產生出我們欲探討的環狀LTIIb-B5，我們使用了重組蛋白中的內含肽其剪接化學鍵的功能，藉此設計出有效的環狀蛋白黏膜佐劑。接著進行Wildtype和環狀LTIIb-B5的結構差異性比較，本篇研究使用的實驗方法包括MALDI-TOF-MS、分析型超高速離心(AUC)、核磁共振技術(NMR)以及Bio-SAXS和X射線繞射。在數個實驗中我們都證實環狀LTIIb-B5如同Wildtype具有五聚體的結構，其中在 X 射線所分析出的五聚體中能提供我們一項更為詳細的訊息:LTIIb-B5在骨架環化後結構並沒有太大的改變。重要的是環狀LTIIb-B5在高溫環境的NMR量測下具有良好耐熱性，且 1H15N 二維光譜滴定實驗顯示其與下游的醣類受體GD1a有相互作用，此作用力證據能與我們在晶體結構上所觀察到的醣類結合口袋相互佐證。
我們期望環狀LTIIb-B5能夠改善它們在生物學上的應用，例如增加在體內的半衰期，或是提升與上下游受體的結合力，進而提高LTIIb-B5佐劑活性的強度。因此在這篇論文中，我們提供內含肽介導環化作用提升LTIIb-B5穩定性的證明，期望未來環狀LTIIb-B5有利於人體疫苗開發上及廣泛應用於相關領域中。

Intein shows ability to ligate polypeptide backbone. The same enzyme mechanism could be extended to cyclize peptides and proteins. As the previous reports, the cyclized proteins contained increased thermo-resistance in solution. The employment of intein in protein
engineering becomes an attractive strategy. In our study, we consider to engineer a potent mucosal protein adjuvant by using the B subunit of type II heat labile enterotoxin (LTIIb-B5) as a target. A strategy to create a cyclic LTIIb-B5 was developed by incorporating the Intein
protein scaffold. The LTIIb-B5 was successfully cyclized during the expression. To realize the structural similarities and differences between wildtype and cyclic LTIIb-B5, the experimental methods including MALDI-TOF-MS, Analytical Ultracentrifugation, Nuclear Magnetic Resonance, Bio-SAXS and X-ray diffraction were used. The experiments consistently conclude the same conformation of P5 symmetry as identified in wildtype LTIIbB5. The X-ray structure further revealed the details of the pentameric structure, indicating no significant change caused by the backbone cyclization. Most importantly, the cyclic protein not only showed good thermal stability during the high-temperature NMR measurement, but also demonstrated the binding to its downstream ligand, GD1a. The result was supported by the similar carbohydrate binding pockets on cyclic LTIIb-B5 surface. Thus, we expect the physical property of cyclic LTIIb-B5 may improve its biological applications, such as elongating half-life in the body or better binding between the upstream and downstream receptors, thereby improving the potential of being the protein adjuvant. Here, we prove a concept of creating stable LTIIb-B5 by cyclization. The study might benefit the development of human vaccines and hopefully be used in related fields in the future.

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