腫瘤壞死因子受體結合因子6 (Tumor-necrosis factor receptor associated factor 6)純化與結晶

Purification and Crystallization of the N-terminal domain of Mouse Tumor-necrosis factor receptor associated factor 6

饒若卉(Jo-Hui Jao)

指導教授 ： 蔡麗珠 ； 郭呈欽

國立臺北科技大學/工程學院/有機高分子研究所/碩士(2008年)

摘要

全長為530 個胺基酸的老鼠TRAF6 (Tumor-necrosis factor receptor-associatedfactor 6) 蛋白質為TRAF family 的第六號成員，是 IL-1 (Interlukin-1 receptor) / TLR (Toll-like receptor) 誘導活化真核轉錄因子NF-κB (Nuclear factor κB) 這條路徑中重要的連接蛋白。TRAF6 蛋白質主要有兩個功能區域，一為包含環指 (RING finger)及數個鋅指 (zinc finger)結構的N 端與另一為TRAF family 特有保守序列的C端。目前已知TRAF6 之TRAF-C 端區域的結晶結構，但對於TRAF6-N 端的結晶結構至今尚未有結果發表。為了更進一步瞭解TRAF6 如何與上下游的其他蛋白質氨基酸接觸並進行信息傳遞，本實驗以解析出TRAF6 全長與TRAF-N 端區域結構為目的。將TRAF6 及TRAF6 N 端1-350 胺基酸區域對應之DNA 序列 (以下稱為TRAF6N) 構築在蛋白質表現載體pGEX4T1 上，以大腸桿菌作為宿主系統大量表現TRAF6 與TRAF6N，破菌後發現TRAF6 全長蛋白質全部存在於不水溶的inclusion body，而TRAF6N 則是能得到超過總表現量70%的水溶性蛋白質。TRAF6N 最適純化條件為加入30%硫酸胺 (ammonium sulfate) 使TRAF6N 蛋白質鹽析產生沉澱，再利用GST (glutathione-S-transferase) 親和性管柱進行親和性層析純化後，將所得到之純質TRAF6N 濃縮至濃度為5.2 mg/ml，並以懸滴式蒸氣擴散法進行結晶實驗。

關鍵字

TRAF6 ； 蛋白質純化 ； 蛋白質結晶

並列摘要

Tumor-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is an adaptor protein involved in interleukin-1 receptor (IL-1) and toll-like receptor (TLR) induced activation of nuclear factor κB (NF-kB). TRAF6 contains two domains, C- and N-terminal domains, which associate with other adaptor proteins of IL-1R/TLR pathway. Crystal structure of TRAF6 C-domain has been reported but lack of N-domain which contains a RING finger domain and several zinc finger motifs. In order to understand the protein-protein interaction, we plan to determine crystal structures of the TRAF6 N-domain residues 1~350 (TRAF6N), and the full-length TRAF6. The aim of this study was to clone DNA, protein expression, purification, and crystallization. Our results show that the E. coli expression of TRAF6N and TRAF are in suspension (70%) and
inclusion body, respectively. Recombinant TRAF6N was purified by 30% ammonium sulfate salt-out method and followed a glutathione-S-transferase (GST)-affinity chromatography. TRAF6N protein was concentrated to 5.2 mg/ml and crystallized by the hanging-drop vapor diffusion method.

並列關鍵字

TRAF6 ； protein purification ； protein crystallization

參考文獻

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