Open AccessAmplification of protein expression in a cell free system
Author(s) -
Edgard Resto,
Akira Lida,
M D Van Cleve,
Sidney M. Hecht
Publication year - 1992
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/20.22.5979
Subject(s) - biology , t7 rna polymerase , transcription (linguistics) , microbiology and biotechnology , dihydrofolate reductase , protein biosynthesis , gene , dna , biochemistry , cell free system , gene expression , rna polymerase , plasmid , reticulocyte , rna , enzyme , linguistics , philosophy , escherichia coli , bacteriophage
Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNAfol, 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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