Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway
Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway
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DOI：
10.1110/ps.051902606
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发表时间：
2006-03-01
期刊：
影响因子：
8
通讯作者：
DeLisa, MP
中科院分区：
文献类型：
--
作者：
Fisher, AC;Kim, W;DeLisa, MP
结构基因组学工作和生物技术企业面临的最棘手的问题之一是由于折叠不良和不溶性而无法有效地生产功能蛋白质。此外，蛋白质错误折叠和聚集与许多人类疾病有关，如阿尔茨海默氏症。因此，允许直接监测、操纵和改善蛋白质折叠的稳健的细胞测定可能具有深远的影响。我们报告了在活细菌细胞中蛋白质折叠和溶解度的遗传选择的发展和表征。该试验的基础是观察到通过细菌双精氨酸易位（达特）途径的蛋白质转运取决于转运前蛋白质的正确折叠。在该系统中，测试蛋白表达为N-末端达特信号肽和C-末端TEM 1 β-内酰胺酶报告蛋白之间的三联融合体。我们证明了大肠杆菌细胞在表达Tat靶向测试蛋白/β-内酰胺酶融合的选择性培养基上的存活与测试蛋白的溶解度相关。使用该测定，我们从A β 42序列的大型组合文库中分离出阿尔茨海默氏A β 42肽的溶解度增强变体，从而证实我们的测定是可溶性蛋白质的高效选择工具。通过允许细菌达特途径对表达的靶蛋白序列施加折叠质量控制，我们已经产生了用于监测活细胞中的蛋白质折叠和溶解度、用于溶解度增强蛋白质的分子工程或用于分离稳定聚集倾向蛋白质的因子和/或细胞条件的有力工具。
One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's A beta 42 peptide from a large combinatorial library of A beta 42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.