Isolation of intracellular proteinase inhibitors derived from designed ankyrin repeat proteins by genetic screening
Isolation of intracellular proteinase inhibitors derived from designed ankyrin repeat proteins by genetic screening
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DOI：
10.1074/jbc.m602506200
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发表时间：
2006-12-29
影响因子：
4.8
通讯作者：
Plueckthun, Andreas
中科院分区：
文献类型：
--
作者：
Kawe, Martin;Forrer, Patrik;Plueckthun, Andreas
在蛋白质水平上对蛋白质活性的特异性细胞内抑制是验证或调节细胞过程的非常有价值的工具。我们在这里展示了设计的锚蛋白重复蛋白（DARPins）作为定制的细胞内蛋白酶抑制剂的使用。位点特异性蛋白水解过程在许多生物过程的调控中起着至关重要的作用，从基本的细胞功能到病毒的繁殖。烟草侵染病毒是一种重要的植物病原体，它的NIa（pro）蛋白酶在大肠杆菌中可以功能表达而不伤害细菌。为了确定这种蛋白酶的抑制剂，我们首先从DARPins组合文库中选择了与之结合的结合物，并用一种新的体内蛋白酶抑制筛选方法测试了这种结合物。为此，一个由大肠杆菌RNA聚合酶的omega亚基组成的杂交蛋白被共价融合到一个dna结合蛋白，lambda cI抑制蛋白，在这两个蛋白之间的连接物中含有一个NIa（pro）切割位点。因此，这种转录激活因子被位点特异性蛋白水解裂解失活，而这种裂解的抑制剂可以通过报告基因的转录重组来鉴定。按照这两步选择和筛选方法，我们可以从高度多样化的组合DARPin文库中快速分离出细胞内活性的NIapro蛋白酶抑制剂。这些发现强调了DARPins在细胞内空间调节蛋白质功能方面的巨大潜力。此外，我们的新基因筛选可以帮助选择和鉴定基于其他蛋白质支架甚至低分子量化合物的定制蛋白酶抑制剂。
The specific intracellular inhibition of protein activity at the protein level is a highly valuable tool for the validation or modulation of cellular processes. We demonstrate here the use of designed ankyrin repeat proteins (DARPins) as tailor-made intracellular proteinase inhibitors. Site-specific proteolytic processing plays a critical role in the regulation of many biological processes, ranging from basic cellular functions to the propagation of viruses. The NIa(pro) proteinase of tobacco etch virus, a major plant pathogen, can be functionally expressed in Escherichia coli without harming the bacterium. To identify inhibitors of this proteinase, we first selected binders to it from combinatorial libraries of DARPins and tested this pool with a novel in vivo screen for proteinase inhibition. For this purpose, a hybrid protein consisting of the omega subunit of E. coli RNA polymerase was covalently fused to a DNA-binding protein, the lambda cI repressor, containing an NIa(pro) cleavage site in the linker between the two proteins. Thus, this transcriptional activator is inactivated by site-specific proteolytic cleavage, and inhibitors of this cleavage can be identified by the reconstitution of transcription of a reporter gene. Following this two-step approach of selection and screening, we could rapidly isolate NIapro proteinase inhibitors active inside the cell from highly diverse combinatorial DARPin libraries. These findings underline the great potential of DARPins for modulation of protein functionality in the intracellular space. In addition, our novel genetic screen can help to select and identify tailor-made proteinase inhibitors based on other protein scaffolds or even on low molecular weight compounds.