NOVEL TECHNOLOGY FOR THE ANALYSIS OF GENE EXPRESSION

该项目的目标是开发新技术，用于快速筛选体外表达的蛋白质，以及与癌症相关的特定基因。标记物和可光裂解的接头将在无细胞合成过程中与特别制备的错氨酰化tRNA结合到蛋白质中。与常规放射性方法相比，将高荧光氨基酸掺入新生蛋白质中可以显著减少检测和表征此类蛋白质所需的时间，并且适合于高通量自动化。将专有的光可切割接头掺入新生蛋白质中有助于通过各种技术（包括凝胶位移测定、质谱和毛细管电泳）进行基于亲和力的快速纯化和下游物理和功能分析。这种方法将在第一阶段通过将各种荧光标记物和光可裂解的连接体掺入四种模型蛋白质中，通过分析这些修饰蛋白质的性质以及通过测试用于分离和检测这些蛋白质的各种方法来进行评估。该技术将在临床水平上进行评估，以在第一阶段对APC基因中的链终止突变进行基因诊断，并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中具有商业应用。产品包括试剂、用于将荧光标记物和PC-连接物整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，如蛋白质截短试验（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).