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Nucleic Acids Research, 1992, Vol. 20, No. 22 5979-5983
© 1992

METHODS

Amplification of protein expression in a cell free system

Edgard Resto, Akira lida, Mark D.Van Cleve and Sidney M. Hecht*

Departments of Chemistry and Biology, University of Virginia Charlottesville, VA 22901, USA

* To whom correspondence should be addressed

Received August 19, 1992. Accepted October 21, 1992.

Large quantities of a catalytically active protein have been produced in a cell free system. More than 109 copies of protein were produced from each DNA plasmid containing DNAfo/i the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polymerase chain reaction (PCR) and (ii) in vitro RNA transcription by T7 RNA polymerase, followed by (iii) translation of the run-off transcripts in a rabbit reticulocyte system. The protein product had the expected size (18 kDa) and catalyzed the NADPH-dependent reduction of 7,8-dihydrofolic acid to 5,6,7,8-tetrahydrofolic acid as efficiently as authentic DHFR. Potential applications of the strategy include large scale production of enzymes containing synthetic amino acids and facilitation of the characterization of the function of genes encountered in genomic mapping studies.


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