TY - JOUR
T1 - Amplification of protein expression in a cell free system
AU - Resto, Edgard
AU - Lida, Akira
AU - Cleve, Mark D.van
AU - Hecht, Sidney M.
N1 - Funding Information:
We thank Dr. John Abelson and Mr. George Komatsoulis, California Institute of Technology, for the plasmid containing DNAjro/. This work was supported by NIH Research Grant GM43328, and by an NIH Postdoctoral Fellowship (GM13651) to E.R.
PY - 1992/11/25
Y1 - 1992/11/25
N2 - 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.
AB - 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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U2 - 10.1093/nar/20.22.5979
DO - 10.1093/nar/20.22.5979
M3 - Article
C2 - 1281316
AN - SCOPUS:0026482587
VL - 20
SP - 5979
EP - 5983
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 22
ER -