NOVEL TECHNOLOGY FOR THE ANALYSIS OF GENE EXPRESSION

该项目的目标是开发快速筛选体外表达的、来自与癌症相关的特定基因的蛋白质的新技术。在特殊准备的氨基酰化tRNAs的无细胞合成过程中，标志物和可光解连接物将被结合到蛋白质中。与传统的放射性方法相比，在新生蛋白质中掺入高荧光氨基酸可以显著减少检测和鉴定这类蛋白质所需的时间，并且易于实现高通量自动化。在新生蛋白质中引入专有的光可裂解连接体，有助于通过凝胶位移分析、质谱分析和毛细管电泳法等多种技术快速亲和纯化和下游物理和功能分析。这一方法将在第一阶段进行评估，将各种荧光标记和可光切割的连接物加入到四个模型蛋白质中，分析这些修饰蛋白质的性质，并测试这些蛋白质的分离和检测的各种方法。这项技术将在临床层面上进行评估，在第一阶段对APC基因链终止突变进行基因诊断，在第二阶段将扩展到检测与其他癌症综合征相关的各种基因突变。拟议的商业应用：开发快速检测和分离无细胞产生的蛋白质的方法将在癌症研究和临床诊断的许多领域获得商业应用。产品包括试剂、将荧光标记和PC连接物结合到新生蛋白质中的试剂盒，以及用于筛选遗传缺陷的自动化系统，如蛋白质截断测试(PTT)。

The objective of this project is to develop new technology for the rapid screening of proteins that are expressed in vitro and derived from specific genes linked to cancer. Markers and photo cleavable linkers will be incorporated into proteins during cell-free synthesis with specially prepared misaminoacylated tRNAs. The incorporation of highly fluorescent amino acids into nascent proteins can significantly decrease the time needed to detect and characterize such proteins compared with conventional radioactive methods and is amenable to high throughput automation. The incorporation of proprietary photo cleavable linkers into nascent proteins facilitates their rapid affinity-based purification and downstream physical and functional analysis by a variety of techniques including gel-shift assays, mass spectrometry and capillary electrophoresis. This approach will be evaluated during Phase I by incorporating a variety of fluorescent markers and photo cleavable linkers into four model proteins, by analyzing the properties of these modified proteins and by testing various methods for the isolation and detection of these proteins. This technology will be evaluated at the clinical level for genetic diagnosis of chain terminating mutations in the APC gene during Phase I, and in Phase II will be extended to detect a variety of genetic mutations linked to other cancer syndromes. PROPOSED COMMERCIAL APPLICATIONS: The development of rapid methods for the detection and isolation of cell- free produced proteins will have commercial applications in many areas of cancer research and clinical diagnostics. Products include reagents, kits for incorporating fluorescent markers and PC-linkers into nascent proteins, and automated systems for the screening of genetic defects such as the protein truncation test (PTT).