酵母朊蛋白Sup35NM体外淀粉样纤维的形成动态研究  

Dynamics of in vitro amyloid fiber formation of yeast prion protein Sup35NM

作　　者：魏海燕[1] 刘英霞[2] 王健伟[3] 屈建国[3] 赵蔚明[1] 于修平[1] 洪涛[1] 

机构地区：[1]山东大学医学院病原生物学研究所,济南山东大学与250012 [2]中国医学科学院基础医学研究所 [3]中国疾病预防控制中心病毒病预防控制所洪涛院士实验室

出　　处：《中华实验和临床病毒学杂志》2006年第1期39-42,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金重大项目(30291000)

摘　　要：目的研究酵母朊蛋白Sup35在近似于生理环境的体外条件下淀粉样纤维形成的动态过程,为阐明淀粉样纤维的形成机制提供材料和线索。方法在E.coli中表达Sup35蛋白的NM段,并用Ni2+螯合层析对重组蛋白在变性条件下进行纯化,在不同的时间点用透射电子显微镜观察、圆二色谱检测以及蛋白酶K消化试验,同时利用硫黄素(thioflavin T,ThT)结合试验对淀粉样纤维形成的动态过程进行检测。结果在变性条件下成功纯化出Sup35NM重组蛋白。利用透射电子显微镜观察到了Sup35NM蛋白在PBS(pH7·4)缓冲液中发生聚集时的形态变化,圆二色谱检测显示该过程伴随蛋白结构由α-螺旋到β-折叠的转变。纤维具有较强的抗蛋白酶K消化的特性。ThT结合试验提示淀粉样纤维的形成在经过了一个快速的上升阶段后达到平台期,纤维形成的速率会随着蛋白浓度的提高而加快。结论酵母朊蛋白Sup35NM在接近生理环境的体外条件下极易发生聚集,聚集物具有淀粉样纤维性质,Sup35NM淀粉样纤维形成的动态过程为淀粉样变成核聚集模型的研究提供了依据。Objective To investigate the dynamics of amyloid fiber formation of yeast （ Saccharomyces cerevisiae） prion protein Sup35NM under the native condition to provide materials and clues for the elucidation of amyloid fiber formation. Methods The Sup35NM gene was cloned and expressed in E. coli. The recombinant Sup35NM protein was purified under denaturing conditions through Nickel-Sepharose chromatography. Aliquots were removed at designated time points for transmission electron microscopy （TEM）, circular dichroism （CD） spectm,protease K resistance assay, as well as thioflavin T （ThT） binding assay. Results The Sup35NM expressed and purified under denaturing conditions. The morphological alteration of the Sup35NM in PBS （pit7.4） during the protein aggregation and amyloid fiber formation was visualized by TEM. The CD assay showed that the course of amyloid fiber formation underwent a conformational shift from α-helix to β-sheet. The fibers had higher capacity of resistance to protease K digestion compared to the monomers. ThT fluorescence assay displayed a rapid growth phase before reaching a final equilibrium phase during the fiber formation, and the higher concentration of Sup35NM could greatly accelerate the fiber formation in vitro. Conclusion Yeast prion protein Sup35NM forms amyloid readily under native conditions in vitro. The dynamics of Sup35NM amyloid formation may provide supporting evidences for the nucleating polymerization models of arnyloid fiber formation.

关 键 词：淀粉样蛋白 朊病毒 酵母菌 

分 类 号：R363[医药卫生—病理学]