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標題: 一、果蠅Hopscotch kinase domain蛋白結晶與結構預測分析 二、產酸克雷白氏菌Klebsiella oxytoca之Fosfomycin resistance protein FosA的蛋白表現、純化與結晶
A. Crystallization and Structure Prediction of Hopscotch Kinase Domain from Drosophila melanogaster B. Expression, Purification and Crystallization of Fosfomycin Resistance Protein FosA from Klebsiella oxytoca
作者: 李佳燁
Lee, Jia-Ye
關鍵字: JAK/STAT訊息傳遞;JAK/STAT signaling pathway;果蠅;Hopscotch;產酸克雷白氏菌;FosA;Drosophila melanogaster;Hopscotch;Klebsiella oxytoca;FosA
出版社: 基因體暨生物資訊學研究所
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一、果蠅Hopscotch kinase domain蛋白結晶與結構預測分析

JAK/STAT訊息傳導路徑是細胞重要的調控機制,對於細胞增生、維持恆定、免疫反應與幹細胞維持扮演重要的角色,此路徑異常與許多癌症發生有關。Janus kinases (Jaks)為非受體型的酪胺酸激酶,它會將signal transducers and activators of transcription(STATs)磷酸化,以啟動轉錄作用。在哺乳動物的JAK/STAT路徑中,有四個Jaks與七個STATs,而在果蠅的JAK/STAT系統中,只有一個Jak kinase—Hopscotch (HOP)與一個轉錄因子STAT92E。由於果蠅與哺乳動物JAK/STAT訊息傳遞鍊在演化上具有高度保留性,而果蠅系統相對簡化的特色,使得果蠅成為研究JAK/STAT路徑良好的模式生物。由於目前尚未有Hopscotch的結構資訊,本實驗選殖黑腹果蠅(Drosophila melanogaster) HOP的kinase domain,預期以蛋白結晶方式解析其分子結構。目前已可成功純化出水溶性良好之HOP kinase domain,未來將持續進行結晶條件篩選。我同時以生物資訊方法,先預測出HOP kinase domain的結構,並探討其是否可能與FERM domain交互作用。我也利用分子對接預測研究kinase domain與其抑制劑的結合方式。這些結果可以幫助我們更加了解HOP蛋白結構與功能之間的關係。

二、產酸克雷白氏菌Klebsiella oxytoca之Fosfomycin resistance protein, FosA的蛋白表現、純化與結晶
Fosfomycin是自然界中廣泛存在的抗生素,對於革蘭氏陽性菌與革蘭氏陰性菌皆有作用,目前臨床上用於治療非複雜型泌尿道感染。Fosfomycin藉由干擾細菌細胞壁合成來抑制細菌的生長。然而細菌的抗藥性有逐漸增加的趨勢,例如肺炎克雷白氏菌是造成嚴重院內感染的菌種之一,其產生的Fosfomycin resistance protein(FosA)會將fosfomycin接上一個glutathione,使fosfomycin失去活性。FosA存在於許多微生物基因體中,然而目前在蛋白結構資料庫內,僅有綠膿桿菌(Pseudomonas aeruginosa) FosA結構資訊,因此,本實驗室將產酸克雷白氏菌E718的FosA利用E. coli進行蛋白質表現,篩選出具有fosfomycin抗性的菌株,並已純化出水溶性良好並且具有功能之FosA,進行蛋白結晶試驗。未來仍須持續調整結晶條件以得到蛋白質晶體。

A. Crystallization and Structure Prediction of Hopscotch Kinase Domain from Drosophila melanogaster

JAK/STAT signaling pathway is an essential component regulating cell proliferation, homeostasis, immune response and stem-cell maintenance. Misregulation of the pathway is associated with multiple human malignancies such as leukemia and solid tumors. Janus kinases (Jaks) are non-receptor tyrosine kinases which are activated upon autophosphorylation. Activated Jaks then phosphorylate associated receptors and STATs, resulting in STATs translocation to the nucleus where they bind to target DNA sequence to activate transcription. In mammals, there are four Jaks and seven STATs. Whereas in Drosophila, there is only one Jak kinase known as Hopscotch (HOP) and a transcription factor STAT92E. A simpler pathway of Drosophila JAK/STAT and high level of conservation between insects and mammals make Drosophila an excellent model for studying JAK/STAT pathway. Detailed structural information on Jaks is needed to understand the mechanism of Jak activation. Therefore, we expressed and purified the kinase domain of HOP from Drosophila melanogaster for crystallization experiments. In addition, we predicted the structure of HOP kinase domain and its interaction with FERM domain, and performed docking studies with possible inhibitors. These results will improve our understanding into the structure-function relationship of this important protein.

B. Expression, Purification and Crystallization of Fosfomycin Resistance Protein, FosA from Klebsiella oxytoca

Fosfomycin is a natural product which has antimicrobial activities against both Gram-positive and Gram-negative bacteria. Fosfomycin inhibits bacterial growth by interfering with the first committed step in microbial cell wall biosynthesis. This antibiotic is currently used for treatment of acute uncomplicated lower urinary tract infection. However, after the use of fosfomycin in the clinic, resistance to the antibiotic began to emerge. Fosfomycin resistance protein, FosA, a 16 kDa polypeptide is responsible for the addition of glutathione (GSH) to the antibiotic, rendering it inactive. To date, structural information of FosA is limited to that encoded in Pseudomonas aeruginosa. We have generated a soluble and functional recombinant FosA from Klebsiella oxytoca E718 and carried out randomized protein crystallization trials. It is important to further improve condition that favors protein crystals formation.
其他識別: U0005-2608201316263200
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