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標題: Xanthomonas campestris pv. Campestris重金屬耐受性蛋白XC2981的大量表達和結構解析
Expression and structural analysis of a divalent cation tolerance protein XC2981 from Xanthomonas campestris pv. Campestris
作者: 林建宏
Lin, Chien-Hung
關鍵字: XC2981;Xanthomonas campestris
出版社: 生物化學研究所
Xanthomonas campestris pv. campestris (Xcc) 為格蘭氏陰性菌,會感染十字花科植物,造成農業上重大的損失。XCC會生成多醣體 (Xanthan),在工業上的應用相當廣泛。因此,我們將以X.campestris pv. campestris str. 17為研究素材,利用X-ray晶體繞射的方法解析其內生性蛋白質的三級結構,並利用解析出的結構研究XCC的生物機制與調控。X.campestris pv. campestris str. 17 目前已由台灣團隊完成定序及基因註解。利用PCR截取欲解析蛋白質的基因,並大量表現後,藉由X-ray的晶體繞射,分析收集到的繞射點強度,即可解析出欲求得的蛋白質三級結構。
由X.campestris pv. campestris str. 17 的基因註解和序列的比對,挑選了分別和重金屬有交互作用與可調控重金屬的吸收利用的 XC2981 與 XC3379 作為研究的目標。XC2981 根據序列比對,發現從簡單的原核生物到高等的真生物,皆存在類似的蛋白質。並發現當在這些相似蛋白質中,若有胺基酸產生突變,生物體對於二價重金屬的容忍度就會降低,甚至造成死亡。因此推測 XC2981 可能和二價重金屬的吸收或控制有關,但尚未有詳細的機制被發表。目前已經成功的收集到解析度 2.6Å 的原生蛋白 (Native protein) 繞射光譜,也成功的收到Se-Met的繞射光譜,但卻無法解析出XC2981的相位,現在已經製備好共結晶的蛋白,並嘗試收集其繞射光譜。
XC3379 經序列比對,推測可能是 Ferric uptake regulator,其會辨認和調控鐵離子相關蛋白質的啟動子中的特定序列,稱之為Fur box。隨著環境中鐵離子的濃度變化,Ferric uptake regulator會調控相關蛋白質的表現,以達到利用與調控鐵離子濃度的目的。XC3379 已經能大量表達,但尚在尋找合適的結晶條件。

Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. Therefore, X.campestris pv. campestris str. 17 is chosen for our research. At present, DNA sequences of X.campestris pv. campestris str. 17 have been completely determined. By means of these sequences and through bioinformatics approach, we can discover interesting genes that can be amplified by PCR. Then, optimized conditions could be screened to obtain high purity and large amount of desired proteins. Most importantly, the tertiary structures of the desired proteins could be solved by the powerful X-ray diffraction tool.
Several genes correlating with heavy metal ion tolerance were studied in this thesis. XC2981 is deduced as a divalent cation tolerance protein from a bioinformatics study.. From prior molecular genetics studies on the E. coli cutA locus, it was revealed that some mutations in this locus can lead to copper sensitivity due to its increased uptake. Hence CutA is believed to participate in copper ion tolerance in E. coli. We have successfully expressed XC2981 in E. coli in high yield. X-ray diffraction data of native XC2981 crystal diffracted to a resolution of 2.6 has been collected. However, the phase could not be determined, possibly due to the fact that XC2981 contains only one Se-Met at the N-terminal end. Cocrystallization of XC2981 with heavy metal is another alternative and will be prepared for X-ray diffraction and phase determination.
XC3379 is deduced as a ferric uptake regulator (Fur), which can regulate the expression of proteins relative to the uptake of iron by binding with the respective promoter. XC3379 has also been successfully expressed in high yield. However, the best condition for its crystallization is yet to be found.
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