Amplification of protein expression in a cell free system

Edgard Resto, Akira Iida, Mark D. Van Cleve, Sidney Hecht

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)5979-5983
Number of pages5
JournalNucleic Acids Research
Volume20
Issue number22
StatePublished - Nov 25 1992
Externally publishedYes

Fingerprint

Cell-Free System
Amplification
Tetrahydrofolate Dehydrogenase
Transcription
Gene
Proteins
Protein
RNA
Cell
DNA
Bacterial Genes
Gene encoding
Polymerase Chain Reaction
Acids
Gene Amplification
Reticulocytes
Polymerase chain reaction
DNA-Directed DNA Polymerase
Rabbit
NADP

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

Resto, E., Iida, A., Van Cleve, M. D., & Hecht, S. (1992). Amplification of protein expression in a cell free system. Nucleic Acids Research, 20(22), 5979-5983.

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 journalArticle

Resto, E, Iida, A, Van Cleve, MD & Hecht, S 1992, 'Amplification of protein expression in a cell free system', Nucleic Acids Research, vol. 20, no. 22, pp. 5979-5983.
Resto E, Iida A, Van Cleve MD, Hecht S. Amplification of protein expression in a cell free system. Nucleic Acids Research. 1992 Nov 25;20(22):5979-5983.
Resto, Edgard ; Iida, Akira ; Van Cleve, Mark D. ; Hecht, Sidney. / Amplification of protein expression in a cell free system. In: Nucleic Acids Research. 1992 ; Vol. 20, No. 22. pp. 5979-5983.
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