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

双向凝胶电泳(2D-Gel)是一种能够 在一块凝胶上分离数千种多肽。它的用处在于 然而，受限于生物化学鉴定的困难 蛋白质在凝胶上分离，尤其是较小的蛋白质。我们是 参与开发一种方法，如果成功，将使其 可以使用2D-Gel选择编码特定基因的cDNA克隆 多肽。基本的方法是首先从大鼠肝脏中分离出mRNA 上皮(RLE)细胞，逆转录并克隆后续的 插入到lambda-ECC噬菌体文库中，设计成 可以对重组子进行正向选择。将该基因插入到一个 T7启动子和终止子，使其能够在体外转录成 产生ERSATZ信使核糖核酸，理想情况下，它是对 文库来源的真正的mRNAs。这件事的复杂性 库可以通过在翻译产品上运行2D-Gel来访问 代谢物m RNA。可以通过以下方法分离特定多肽的克隆 按顺序细分库并降低其复杂性，直到 只剩下一个负责特定地点的克隆人。我们有 比较从非反式病毒中提取的真mRNA的2D-凝胶图谱 形成RLE细胞和v-Ha-ras癌基因转化的细胞。这个 模式非常相似，但注意到一些始终如一的差异， 尤其是RAS中两个约15 kDa的斑点 细胞。构建了Lambda-ECC的cDNA文库，并进行了以下工作 目前关注的是准确表达和翻译的条件-- 逆转录酶基因的表达。

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.