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Proteolytic processing of the replicase polyprotein of Grapevine rupestris stem pitting-associated virus.

机译:蛋白水解加工葡萄干茎凹坑相关病毒的复制酶多蛋白。

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摘要

A region of ORF 1 from Grapevine rupestris stem pitting-associated virus was expressed in Escherichia coli, baculovirus, and in vitro transcription/translation expression systems to test its putative proteolytic activity. The region expressed included the predicted protein domains for an ovarian tumour-like protease, papain-like protease, helicase, and RNA-dependant RNA polymerase. A protein equivalent to the theoretical size of the cloned region was expressed in the E. coli and baculovirus systems. The protein produced in these systems was largely insoluble and likely inactive. The activity or cleavage sites of the putative proteases could not be determined from either system by Western analysis. Knock-outs of the predicted active sites of either or both protease domains showed no difference in expression profile to the wild-type constructs. Upon review of results obtained from the expression systems, the putative proteases appear to be inactive when expressed in E. coli, or insect cells infected by a recombinant baculovirus.
机译:在大肠杆菌,杆状病毒和体外转录/翻译表达系统中表达了来自葡萄藤茎点蚀相关病毒的ORF 1区域,以测试其假定的蛋白水解活性。表达的区域包括卵巢肿瘤样蛋白酶,木瓜蛋白酶,解旋酶和RNA依赖性RNA聚合酶的预测蛋白结构域。在大肠杆菌和杆状病毒系统中表达了与克隆区域理论大小相等的蛋白质。在这些系统中产生的蛋白质大部分是不溶的,可能是无活性的。推定的蛋白酶的活性或切割位点不能通过Western分析从任一系统中确定。任一或两个蛋白酶结构域的预测活性位点的敲除显示出与野生型构建体的表达谱无差异。根据从表达系统获得的结果,推定的蛋白酶在大肠杆菌或重组杆状病毒感染的昆虫细胞中表达时似乎无活性。

著录项

  • 作者

    Rak, John.;

  • 作者单位

    University of Guelph (Canada).;

  • 授予单位 University of Guelph (Canada).;
  • 学科 Agriculture Plant Pathology.;Biology Virology.
  • 学位 M.Sc.
  • 年度 2009
  • 页码 86 p.
  • 总页数 86
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 植物病理学;
  • 关键词

  • 入库时间 2022-08-17 11:37:38

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