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Expression, purification and crystallization of the C-terminal domain of human ubiquitin specific protease 7.

机译:人泛素特异性蛋白酶7 C末端结构域的表达,纯化和结晶。

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摘要

USP7 is an ubiquitin specific protease. It functions as a deubiquitinating enzyme that removes ubiquitin from target proteins. USP7 regulates the protein levels of p53, Mdm2 and MdmX. It also interacts with FOXO4 and viral proteins EBNA1, ICP0. USP7 has four domains. Two C-terminal domains which interact with ICP0 and FOXO4 are believed important for the substrate recognition, regulation and protein-protein interaction.GST pull down assays were performed to study the interaction between USP7 and ICP0. Three ICP0 peptides showed positive binding with USP7. The peptides share a 7 amino acid sequence (-RKCARKT-). ICP0 615-629 (-PRGP-RKCARKT-RHAE-) showed strongest binding ability. Four positively charged amino acids were found in the seven residues and this may suggest that these amino acids playing a role in the interactionA total of 15 C-terminal protein constructs were studied. They were prepared, expressed and purified for protein crystallization trials in order to determine their three dimensional structures. Protein precipitation, aggregation and degradation problems persisted with the nine soluble proteins and these are negative factors for protein crystallization. Four proteins have been crystallized and more work must be done to improve the crystals.
机译:USP7是泛素特异性蛋白酶。它起脱泛素化酶的作用,可从靶蛋白中去除泛素。 USP7调节p53,Mdm2和MdmX的蛋白质水平。它还与FOXO4和病毒蛋白EBNA1,ICP0相互作用。 USP7有四个域。据信两个与ICP0和FOXO4相互作用的C末端结构域对于底物识别,调控和蛋白质-蛋白质相互作用很重要。进行GST下拉实验研究USP7与ICP0之间的相互作用。三种ICP0肽与USP7呈阳性结合。这些肽共有7个氨基酸序列(-RKCARKT-)。 ICP0 615-629(-PRGP-RKCARKT-RHAE-)显示出最强的结合能力。在七个残基中发现了四个带正电荷的氨基酸,这可能表明这些氨基酸在相互作用中起作用。总共研究了15个C端蛋白质构建体。它们被制备,表达和纯化用于蛋白质结晶试验,以确定它们的三维结构。九种可溶性蛋白质仍然存在蛋白质沉淀,聚集和降解问题,这些是蛋白质结晶的负面因素。四种蛋白质已经结晶,必须做更多的工作来改善晶体。

著录项

  • 作者

    Zheng, Hong.;

  • 作者单位

    York University (Canada).;

  • 授予单位 York University (Canada).;
  • 学科 Biology, Molecular.
  • 学位 M.Sc.
  • 年度 2010
  • 页码 141 p.
  • 总页数 141
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 ;
  • 原文服务方 国家工程技术数字图书馆
  • 关键词

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