Plasmid cDNA-directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system.
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.
Craig
D
D
Department of Biochemistry, University of Cambridge, UK.
Howell
M T
MT
Gibbs
C L
CL
Hunt
T
T
Jackson
R J
RJ
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics*; D005796:Genes; D007438:Introns; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008836:Micrococcal Nuclease; D008969:Molecular Sequence Data; D010957:Plasmids; D011188:Potassium / Q000494:pharmacology; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014982:Xenopus laevis
D015636:Magnesium Chloride; D012313:RNA; D004247:DNA; D008836:Micrococcal Nuclease; D011188:Potassium