title: Amplification of protein expression in a cell free system.
abstract: 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.
mesh_terms: D002474:Cell-Free System; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D012313:RNA; D013762:Tetrahydrofolate Dehydrogenase