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，这些蛋白质来自与癌症相关的特定基因。标记物和光可切割的连接物将在无细胞合成过程中与特殊制备的错胺酰化trna结合到蛋白质中。与传统的放射性方法相比，将高荧光氨基酸掺入新生蛋白质中可以显着减少检测和表征这些蛋白质所需的时间，并且可以实现高通量自动化。将专有的光可切割连接物结合到新生蛋白质中，促进了它们基于亲和的快速纯化和下游物理和功能分析，包括凝胶移位测定、质谱分析和毛细管电泳。该方法将在第一阶段进行评估，通过将各种荧光标记和光可切割连接物结合到四种模型蛋白中，分析这些修饰蛋白的特性，并测试分离和检测这些蛋白的各种方法。该技术将在临床水平上进行评估，用于I期APC基因链终止突变的遗传诊断，并在II期扩展到检测与其他癌症综合征相关的各种基因突变。建议的商业应用：开发快速检测和分离无细胞蛋白的方法将在癌症研究和临床诊断的许多领域具有商业应用。产品包括将荧光标记物和pc连接物结合到新生蛋白质中的试剂、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).