...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >A Cleavage Enzyme-Cytometric Bead Array Provides Biochemical Profiling of Resistance Mutations in HIV-1 Gag and Protease
【24h】

A Cleavage Enzyme-Cytometric Bead Array Provides Biochemical Profiling of Resistance Mutations in HIV-1 Gag and Protease

机译：卵裂酶法测定珠阵列提供抗生突变的HIV-1堵嘴和蛋白酶的生化分析。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Most proteaseu0001substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences.nThese assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influencesnenzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individualsnresistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gagnsubstrate contribute to inhibitor resistance.We have developed a proteaseu0001substrate cleavage assay, termed the cleavage enzymeu0001ncytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gagnsubstrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescencenutilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (kcat/KM) impartednby protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate.Wenshow that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of smallnmolecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzymeu0001substrate pairnand can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymaticnprocessing of substrate.
机译：大多数蛋白酶的底物测定依赖于短的，由天然或修饰的裂解序列组成的合成肽底物。这些测定不足以询问影响酶促裂解的裂解位点远端的天然底物结构的贡献或对天然底物的抑制剂筛选。从对蛋白酶抑制剂不具有抗性的个体获得的HIV-1分离物的最新证据表明，Gagn底物中蛋白酶切割位点远端或周围的突变有助于抑制剂抵抗。我们开发了一种蛋白酶u0001底物裂解测定法，称为裂解酶u0001细胞计数珠阵列（CE- CBA），它依赖于包含嵌入的切割位点的Gag底物的天然结构域。 Gagn底物表达为荧光的报告融合蛋白，通过流式细胞仪检测荧光损失可以追踪底物的裂解。 CE-CBA可以精确测定Gag底物的裂解位点或非裂解位点中蛋白酶和/或gag中蛋白酶抑制剂抗性突变引起的蛋白酶催化效率（kcat / KM）的变化.Wenshow CE-CBA平台可以识别HIV -1蛋白酶存在于细胞提取物中，有助于识别蛋白酶或其底物Gag的小分子抑制剂。而且，CE-CBA可以很容易地适应任何酶对底物的配对，并且可以用来快速提供催化效率的评估以及系统地筛选底物酶促加工的抑制剂。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号