Plasmid cDNA-directed protein synthesis in a coupled eukaryoticin vitrotranscription-translation system

作者：David Wesley Craig, Michael T. Howell, Catherine L. Gibbs, Tim Hunt, Richard Joseph Jackson · 发表于：Nucleic Acids Research · 年份：1992 · DOI：10.1093/nar/20.19.4987 · 被引用次数：76 · 研究领域：RNA and protein synthesis mechanisms、Viral Infections and Immunology Research、Viral Infectious Diseases and Gene Expression in Insects

A system is described in which transcription of cDNA clones by bacteriophage T7 RNA polymerase is coupled to translation in the micrococcal nuclease treated rabbit reticulocyte lysate in a single reaction of coupled transcription-translation. The monovalent and divalent cation requirements for translation are dominant for optimum expression in this coupled system, so that transcription is relatively inefficient. Nevertheless, the use of appropriate DNA concentrations leads to the synthesis of sufficient RNA to saturate the protein synthesis capacity of the system. The fidelity and efficiency of expression in this coupled system are high, and the degree of purification of the plasmid DNA is relatively uncritical. The system therefore offers very considerable advantages for rapid screening of 'mini-preparations' of cDNA plasmid constructs for retention of the correct reading frame and expression of the desired protein product.