DEVELOPMENT OF AN RLE CELL CDNA LIBRARY FOR CELL-FREE EXPRESSION OF PROTEINS

用于蛋白质无细胞表达的 RLE 细胞 CDNA 文库的开发

• 批准号: 3838484
• 负责人: M J MILLER
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3838484
• 关键词: RNA directed DNA polymerase; bacteriophage lambda; cell free system; cell transformation; complementary DNA; epithelium; gene expression; genetic library; genetic promoter element; genetic techniques; genetic transcription; genetic translation; laboratory rat; liver cells; messenger RNA; method development; molecular cloning; oncogenes; protein engineering; proteins; recombinant DNA; tissue /cell culture

Two-dimensional gel electrophoresis (2D-gel) is a technique capable of separating thousands of polypeptides on a single gel. Its usefulness is limited, however, by the difficulty in biochemically identifying the proteins separated on the gels, especially the minor ones. We are engaged in developing a methodology that, if successful, will make it possible to use 2D-gel to select for cDNA clones that code for specific polypeptides. The basic approach is to first isolate mRNA from rat liver epithelial (RLE) cells, reverse transcribe it and clone the subsequent cDNA into a lambda-ecc bacteriophage library designed so that recombinants can be positively selected. The cDNA is inserted between a T7 promoter and terminator which allows it to be transcribed in vitro to produce ersatz mRNA which is, ideally, an accurate representation of the genuine mRNA from which the library is derived. The complexity of the library can be accessed by running 2D-gel on the translation products of the ersatz mRNA. Clones for particular polypeptides can be isolated by sequentially subdividing the library and reducing its complexity until only a single clone responsible for a specific spot is left. We have compared the 2D-gel patterns from genuine mRNA isolated from non-trans- formed RLE cells and cells transformed by the v-Ha-ras oncogene. The patterns are very similar, but some consistent differences were noted, particularly two spots of approximately 15 kDa in the ras transformed cells. Lambda-ecc cDNA libraries have been constructed and work is currently focused on conditions for the accurate expression and transla- tion of the ersatz mRNA.

二维凝胶电泳（2D-gel）是一种能够 在单一凝胶上分离数千种多肽。 其有用性 然而，由于生物化学鉴定的困难， 在凝胶上分离的蛋白质，尤其是次要的蛋白质。 我们 致力于开发一种方法，如果成功的话， 可以使用2D-凝胶来选择编码特异性的cDNA克隆， 多肽。 基本方法是首先从大鼠肝脏中分离mRNA 上皮（RLE）细胞，逆转录它并克隆随后的 将cDNA导入到设计为使 重组体可以被阳性选择。 cDNA插入到一个 T7启动子和终止子，其允许其在体外转录， 理想情况下，它是一个精确的表达， 真正的mRNA来源于该文库。 的复杂性 可以通过在以下翻译产品上运行2D-gel来访问库： ErxmRNA。 特定多肽的克隆可通过以下方法分离： 顺序地细分库并降低其复杂性， 只留下负责特定点的单个克隆。 我们有 比较了从非反式- 形成RLE细胞和由v-Ha-ras癌基因转化的细胞。 的 模式非常相似，但注意到一些一致的差异， 特别是在ras转化的细胞中的两个约15 kDa的点， 细胞 已经构建了λ-ecc cDNA文库， 目前专注于准确表达和翻译的条件， 的mRNA。