泛素 (ubiquitin) 在許多細胞活動中扮演關鍵調控者的角色，ABINs (A20-binding inhibitors of NF-κB protein) 屬於泛素鏈結合蛋白家族，最近報導指出其與泛素及A20交互作用在NF-κB訊息調控路徑中扮演負向調控子的角色。A20是去泛素化酶 (deubiquitinases, DUBs) 的一員，A20藉由剪切NF-κB訊息調控路徑相關蛋白K63鍵結泛素鏈，並參與接上K48鍵結泛素鏈，使之降解，也具有抑制NF-κB訊息路徑活化功能。文獻指出A20、ABIN2、ubiquitin三者的不正常表現會造成人類疾病，例如: 自體免疫疾病和B細胞淋巴瘤。然而關於A20、ABIN2、ubiquitin的蛋白質交互作用相關研究仍然有限且機制尚不清楚，因此有關其蛋白質交互作用及複合物結構值得去探討。在我們的研究中，我們建立不同片段目標重組蛋白並用E. coli系統表達目標蛋白質，再使用親和性層析與粒徑篩析層析法，做更進一步純化。成功表達A20、A20/ ubiquitin蛋白質複合體，而A20與ABIN2與先前文獻預測不同，沒有形成複合體。取得的蛋白質複合物將使用X光晶體繞射儀、小角度X光散射儀去解析複合物結構和瞭解蛋白質間的交互作用，得到更多有關蛋白質複合物結構資訊，將有助於清楚明瞭NF-κB訊息傳遞蛋白質的調控機制。

Ubiquitin plays a pivotal role in regulating many cellular events. An emerging ubiquitin chain binding proteins, ABINs (A20-binding inhibitors of NF-κB proteins) were reported to negatively regulate the NF-κB signaling pathway. A20 is one of deubiquitinases (DUBs), which also plays as a negative regulator in the NF-κB signaling pathway by cleaving K63-linked polyubiquitins (polyUbs) and conjugating K48-linked polyUbs to substrates. Previous results have been shown that abnormal expression of A20, ABIN2 and ubiquitin could cause human diseases, such as B-cell lymphoma and autoimmune diseases. Till now, the study about protein interaction between A20, ABIN2, and ubiquitin is limited and unclear. Therefore, how protein interaction in the A20, ABIN2, and ubiquitin is worth to study. In my study, we used Escherichia coli system to clone and express the A20, ABIN2, and ubiquitin. The A20 alone and A20/ ubiquitin complex were purified by the size exclusion chromatography with fast performance liquid chromatography (FPLC). However, ABIN2 and A20 couldn’t form the complex that was different from the paper prediction. Based on previous results, we also generated the mutant and tried to get different form of A20. If we can get homogeneous protein, the protein complex will be subjected to X-ray crystallography or Small Angle X-ray Scattering studies to determine the structure and understand protein-protein interaction mechanism of the complexes in the NF-κB signaling pathway.

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