Abstract
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 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 5979-5983 |
| Number of pages | 5 |
| Journal | Nucleic Acids Research |
| Volume | 20 |
| Issue number | 22 |
| State | Published - Nov 25 1992 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Genetics
- Statistics, Probability and Uncertainty
- Applied Mathematics
- Health, Toxicology and Mutagenesis
- Toxicology
- Genetics(clinical)
Cite this
Amplification of protein expression in a cell free system. / Resto, Edgard; Iida, Akira; Van Cleve, Mark D.; Hecht, Sidney.
In: Nucleic Acids Research, Vol. 20, No. 22, 25.11.1992, p. 5979-5983.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Amplification of protein expression in a cell free system
AU - Resto, Edgard
AU - Iida, Akira
AU - Van Cleve, Mark D.
AU - Hecht, Sidney
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 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.
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 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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UR - http://www.scopus.com/inward/citedby.url?scp=0026482587&partnerID=8YFLogxK
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 -