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Nucleic Acids Research, 1992, Vol. 20, No. 19 4987-4995
© 1992

MOLECULAR BIOLOGY

Plasmid cDNA-directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system

David Craig, Michael T. Howell+, Catherine L. Gibbs, Tim Hunt+ and Richard J. Jackson*

Department of Biochemistry, University of Cambridge Tennis Court Road, Cambridge CB2 1QW, UK

* To whom correspondence should be addressed

Received August 6, 1992. Revised September 5, 1992. Accepted September 5, 1992.

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.

+ Present address: ICRF, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar EN6 3LD, UK


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