Prevention of Recombinant Protein Aggregation in E.coli

描述（由申请人提供）：该项目的目标是使用一种新的方法来防止重组蛋白在大肠杆菌过表达过程中的错误折叠和聚集。本项目中使用的系统由易于聚集的蛋白与报告蛋白（绿色荧光蛋白（GFP））融合组成，该报告蛋白报告融合蛋白在体内的溶解度。在蛋白质合成过程中，第三种蛋白结合并折叠聚集倾向蛋白，其共同表达可阻止蛋白质聚集，并导致报告蛋白（GFP荧光）信号的明显变化。该系统被用作两个并行工作的筛选工具。第一种方法是通过随机诱变来设计大肠杆菌伴侣，然后进行分子进化。二是利用酵母基因组DMA和哺乳动物cDNA文库构建表达文库。通过在含有聚集倾向蛋白- gfp融合的大肠杆菌细胞中共表达伴侣突变体或真核orf文库，将鉴定出有效阻止特定重组蛋白或重组蛋白家族聚集的伴侣变异体、新型伴侣和折叠伴侣。该项目的成功结果将有助于解决结构基因组学的瓶颈之一，导致生产适当数量的适当折叠蛋白质或蛋白质复合物作为商业产品或结构研究，并揭示重要的蛋白质-蛋白质相互作用。这种相互作用的结构研究可以导致对伴侣辅助蛋白质折叠的进一步理解，特别是蛋白质折叠和聚集。一些神经退行性疾病，如亨廷顿氏病、阿尔茨海默病和朊病毒疾病，其特征是特定蛋白质聚集体的积累。分子伴侣已被证明调节这些蛋白质聚集体的溶解度状态。作为一个长期目标，本项目旨在深入了解这些疾病相关蛋白的聚集机制和预防聚集的方法。

DESCRIPTION (provided by applicant): The goal of the project is to use a novel approach to prevent recombinant protein misfolding and aggregation during overexpression in E. coli. The system used in this project consists of an aggregation-prone protein fused to a reporter protein (green fluorescent protein (GFP)) that reports solubility of the fusion protein in vivo. Co-expression of a third protein that binds and folds the aggregation-prone protein during protein synthesis prevents protein aggregation and results in a visible change in the signal of the reporter protein (GFP fluorescence). This system is used as a screening tool for two parallel efforts. The first is to engineer E. coli chaperones by random mutagenesis, followed by molecular evolution. The second is to construct expression libraries using yeast genomic DMA and mammalian cDNA libraries. By co-expressing libraries of chaperone mutants or eukaryotic ORFs in the E. coli cells containing the aggregation-prone protein-GFP fusion, chaperone variants, novel chaperones and folding partners that are efficient in preventing aggregation of a given recombinant protein or a family of recombinant proteins will be identified. Successful outcome of this project will help to solve one of the bottle-necks of structure genomics, lead to production of suitable amounts of proper folded proteins or protein complexes as commercial products or for structural studies, and reveal important protein-protein interactions. Structural studies of such interactions can lead to further understanding of chaperone-assisted protein folding in particular and protein folding and aggregation in general. Several neurodegenerative diseases, such as Huntington's disease, Alzheimer's disease and prion diseases, are characterized by accumulation of specific protein aggregates. Molecular chaperones have been shown to modulate solubility states of these protein aggregates. As a long-term goal, this project aims to provide insights into the mechanism of the aggregation by these disease-related proteins and the means to prevent aggregation.