涂7体外转录翻译范例.ppt

In vitro transcription and In vitro translation In vitro Transcription In vitro Transcription Plasmid with T3, T7 or SP6 promoters Linearization of plasmid DNA by restriction enzyme In vitro transcription: Plasmid buffer NTP labeled UTP RNA polymerase DNAse digestion, Phenol/Chloroform extraction and RNA precipitataion Advantages of RNA probes: RNA-RNA hybrids are very stable Tissue can be digested with RNase (dsRNA is not digested) after the hybridization reducing the background Higher specific activity compared to oligonucleotides 蛋白质功能研究思路 DNA水平缺失基因,上调下调基因的表达水平对生命过程的影响 分离纯化蛋白质,研究其基本特性,直接在蛋白质水平进行研究 蛋白样品的获得 收集大量的生物材料纯化(材料来源受限) 重组表达的方式(费时费力) 体外表达 (In vitro translation,快速,高通量快速筛选) In vitro translation In vitro translation 体外表达可以避免过量表达对细胞的毒性，或者形成不溶的包含体，以及表达产物在细胞内的降解，因而可以表达一些对细胞具有毒性的或者不稳定、容易被降解的蛋白。减少了蛋白变性复性的困扰。 体外表达还可以在体系内添加特殊的修饰氨基酸或者标记氨基酸，非常灵活，因而在蛋白质折叠和蛋白质功能研究上都有重要应用。 In vitro translation Cell-Free Expression Systems: 1: E. coli lysate tolerance 2: Rabbit reticulocyte: better for large proteins 3: Wheat germ extract: better for smaller proteins cheaper than rabbit reticulocyte All are prepared as crude extracts containing all the macromolecular components: 70S or 80S ribosomes tRNAs, aminoacyl-tRNA synthetases initiation, elongation and termination factors, etc. To ensure efficient translation, each extract must be supplemented with: amino acids energy sources (ATP, GTP) energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate) other co-factors (Mg2+, K+, etc.). 真核 VS 原核 满足启动子和核糖体结合位点 E. coli lysate 对大肠杆菌蛋白合成机制十分了解,成本低,不需加帽 简单高效,可偶连体外转录和翻译,不需分步优化条件 耐受性(在含有其它不同成分杂质时仍可正常工作) 加入特殊添加剂(蛋白酶抑制剂,分子