NOVEL IN VITRO EXPRESSION METHODS FOR PROTEOME ANALYSIS

蛋白质组学分析是后基因组时代的重要目标。 虽然来自不同生物体的基因的完整列表正在迅速编制，但这些基因中的大多数的功能仍然不确定。这些知识在从药物发现到理解细胞调控的基本机制等领域至关重要。我们将评估一种新的方法，蛋白质组学分析的基础上，在体外表达和蛋白质分离领域的最新进展。到目前为止，还不可能以未改变的形式分离在无细胞提取物中产生的蛋白质。然而，分析来源于组织和单个细胞的mRNA文库的体外表达蛋白质（LIVE-PRO）的功能文库提供了许多优点。通过酶促和化学氨酰化的tRNA掺入专有的光可裂解生物素，通过促进体外表达的蛋白质文库的快速分离，甚至对于具有低拷贝数的蛋白质，解决了这个问题。重要的是，这种方法有利于下游分析和映射的蛋白质组通过各种技术，包括二维凝胶，质谱，亲和结合和生化测定。通过观察对体外表达的蛋白质的不同影响和通过药物-蛋白质相互作用的下游功能分析，将促进药物发现。拟议的商业应用：体外表达蛋白的分离、检测和分析方法的发展将为生物体蛋白质组的定位和鉴定提供新的途径。这些知识对于理解基本的细胞机制和新药的开发都很重要。产品包括用于体外表达蛋白质文库的高通量筛选的试剂盒和系统以及用于药物发现的新方法。请参见随附的Promega支持信。

Proteonomic analysis is an important goal for the post-genome era. While a complete list of genes from different organisms is rapidly being compiled, the function of most of these genes remains undetermined. This knowledge is crucial in areas ranging from drug discovery to understanding the basic mechanisms of cellular regulation. We will evaluate a new approach for proteomic analysis based on recent progress in the area of in vitro expression and isolation of proteins. Until now, it has not been possible to isolate proteins which are produced in a cell- free extract in an unaltered form. However, analysis of functional libraries of in vitro expressed proteins (LIVE-PRO) derived from mRNA libraries of tissues and individual cells offers many advantages. The incorporation of proprietary photocleavable biotins through enzymatically and chemically aminoacylated tRNAs solves this problem by facilitating the rapid isolation of an in vitro expressed protein library, even for proteins with low copy number. Importantly, this approach facilitates the downstream analysis and mapping of the proteome through a variety of techniques including 2D-gels, mass spectrometry, affinity binding and biochemical assays. Drug discovery will be facilitated by observing differential effects on in vitro expressed proteins and by downstream functional analysis of drug-protein interactions. PROPOSED COMMERCIAL APPLICATIONS: The development of new methods for the isolation, detection and analysis of in vitro expressed proteins will provide a new path for mapping and characterizing an organisms proteome. Such knowledge is important for both understanding basic cellular mechanisms and the development of new drugs. Products include kits and systems for high throughput screening of in vitro expressed libraries of proteins and new methodology for drug discovery. See attached support letter from Promega.