Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22907
標題: 一、酵母菌 Rsc8 的結晶研究二、十字花科黑腐病菌第三型作用子 AvrBs1.1、XccA1與 PphE 的結晶研究
1. Crystallization of Rsc8 from Saccharomyces cerevisiae 2. Crystallization of Xanthomonas campestris type III effectors, AvrBs1.1, XccA1 and PphE
作者: 黃慕清
Huang, Mu-Chin
關鍵字: RSC complex;RSC 複合體;Rsc8 subunit;Xcc;Type III effector;RSC8蛋白;十字花科黑腐病菌;第三型分泌系統;第三型作用子
出版社: 生命科學系所
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Acta Crystallogr Sect F Struct Biol Cryst Commun 62(Pt 1): 39-43. Kelley, L. A. and M. J. Sternberg (2009). "Protein structure prediction on the Web: a case study using the Phyre server." Nat Protoc 4(3): 363-71. Mota, L. J. and G. R. Cornelis (2005). "The bacterial injection kit: type III secretion systems." Ann Med 37(4): 234-49. Patel, J. C. and J. E. Galan (2008). "Investigating the function of Rho family GTPases during Salmonella/host cell interactions." Methods Enzymol 439: 145-58. Poirot, O., et al. (2003). "Tcoffee@igs: A web server for computing, evaluating and combining multiple sequence alignments." Nucleic Acids Res 31(13): 3503-6. Rangarajan, E. S., et al. (2008). "Structure and active site residues of PglD, an N-acetyltransferase from the bacillosamine synthetic pathway required for N-glycan synthesis in Campylobacter jejuni." Biochemistry 47(7): 1827-36. Ruth, L., et al. (2000). "alpha-L-iduronidase forms semi-crystalline spherulites with amyloid-like properties." Acta Crystallogr D Biol Crystallogr 56(Pt 4): 524-8. Singer, A. U., et al. (2004). "Crystal structures of the type III effector protein AvrPphF and its chaperone reveal residues required for plant pathogenesis." Structure 12(9): 1669-81. Yagi, N., et al. (2009). "Structure of amyloid fibrils of hen egg white lysozyme studied by microbeam X-ray diffraction." Int J Biol Macromol 45(1): 86-90.
摘要: 
一、酵母菌 Rsc8 的結晶研究

RSC 複合體為酵母菌中非常重要的染色質重組蛋白之一,而 RSC8 在 RSC 複合體中扮演骨架的核心角色。本研究為了解 RSC8 蛋白的功能,將嘗試解析 RSC8 的X光繞射晶體結構。我們以大腸桿菌作為宿主大量表現具有 GST-tag 的 RSC8ΔN 蛋白;此重組蛋白已去除 RSC8 N端的 SWIRM domain。經由親和性液相層析技術純化後,RSC8ΔN 蛋白可以濃縮至 15 mg/ml。目前結晶條件篩選結果發現有幾個條件長出了不規則狀的晶體,但仍無法改善培養出單一晶體來進行 X-ray 繞射解析。目前除了 RSC8ΔN,我們也著手構築 RSC8 上各功能區段之重組蛋白,希望未來可以將各功能區段分別進行結晶結構分析。

二、十字花科黑腐病菌第三型作用子 AvrBs1.1、XccA1與 PphE 的結晶研究

十字花科黑腐病菌 (Xanthomonas campestris pv. campestris, XCC) 可透過其特殊的第三型分泌系統來分泌胞外蛋白,這些分泌蛋白便稱為第三型作用子。推測 XCC 靠著這些作用子蛋白來感染宿主,藉以調控宿主細胞內外之環境以達最適病菌生長之條件。為了進一步了解這些作用子的致病機制,本研究中針對其中三個作用子蛋白 XccA1, AvrBs1.1 和 PphE 進行晶體結構解析,希望可以藉由結構資訊更加瞭解 XCC 與宿主間之交互作用。我們以大腸桿菌大量表現上述三個重組蛋白,可是 XccA1 與 AvrBs1.1 無法以可溶形式存在於大腸桿菌內;PphE 雖然可獲得少量的可溶蛋白,卻容易在純化時被蛋白質水解酶分解為小片段。因此我們將 PphE重新摺疊再進行純化,而後著手 PphE 的結晶條件篩選。目前結晶條件篩選結果顯示,只要化學配方中含有鈣離子,便會快速形成不規則狀晶體,但仍無法改善培養出單一晶體來進行 X-ray 繞射解析。我們目前仍在持續進行結晶條件篩選。

1. Crystallization of Rsc8 from Saccharomyces cerevisiae
RSC is an essential ATP-dependent chromatin remodeling complex from Saccharomyces cerevisiae, and RSC8 is one of the core components of the RSC. To gain insights into the role of Rsc8 in RSC-mediated chromatin remodeling, we tried to determine the crystal structure of RSC8 by using X-ray crystallography. We used Escherichia coli to express the recombinant RSC8ΔN protein, which lacks of N-terminal SWIRM domain and is linked with GST-tag. After purification by affinity column, RSC8ΔN was concentrated to a concentration of 15 mg/ml. During crystallization screening, RSC8ΔN was found forming amorphous crystals in several chemical conditions. However we failed to optimize these conditions, and did not obtain the diffractive crystals. In addition to the crystallization trials on RSC8ΔN, we have constructed recombinant domains of RSC8, and will perform crystallization trials on these truncated proteins.

2. Crystallization of Xanthomonas campestris type III effectors, AvrBs1.1, XccA1 and PphE
Xanthomonas campestris pv. campestris (XCC) uses type III secretion system to deliver effector proteins into host cells. These type III effector proteins function to optimize the host cell environment for bacterial growth. To gain insights into the pathogenic mechanisms of XccA1, AvrBs1.1 and PphE, we tried to determine the crystal structures of these three proteins. We expressed recombinant XccA1, AvrBs1.1 and PphE in Escherichia coil, but XccA1 and AvrBs1.1 were insoluble in cells. PphE was more soluble in E. coli, but it was degraded quickly during purification. Therefore, we purified PphE under a denatured condition and refolded the protein for subsequent crystallization screening. Currently we found amorphous crystals of PphE grown in the chemical conditions containing Ca2+ salts, but did not obtain the diffractive crystals. The crystallization trials are still ongoing.
URI: http://hdl.handle.net/11455/22907
其他識別: U0005-1708200915590100
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