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Titolo:
High-level autoenhanced expression of a single-copy gene in Escherichia coli: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements
Autore:
Marchand, I; Nicholson, AW; Dreyfus, M;
Indirizzi:
Ecole Normale Super, Genet Mol Lab, CNRS, UMR 8541, F-75230 Paris, France Ecole Normale Super Paris France F-75230 UMR 8541, F-75230 Paris, France Wayne State Univ, Dept Biol Sci, Detroit, MI 48202 USA Wayne State Univ Detroit MI USA 48202 ept Biol Sci, Detroit, MI 48202 USA
Titolo Testata:
GENE
fascicolo: 1-2, volume: 262, anno: 2001,
pagine: 231 - 238
SICI:
0378-1119(20010110)262:1-2<231:HAEOAS>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
RNA-POLYMERASE; MESSENGER-RNA; TRANSLATION; SEQUENCE; PROMOTER; PHOSPHORYLATION; SHUTOFF; CLONING; DNA;
Keywords:
protein phosphorylation; E. coli expression system; promoter strength; mRNA stability; translation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
33
Recensione:
Indirizzi per estratti:
Indirizzo: Dreyfus, M Ecole Normale Super, Genet Mol Lab, CNRS, UMR 8541, 46 Rue Ulm,F-75230 Paris, France Ecole Normale Super 46 Rue Ulm Paris France F-75230is, France
Citazione:
I. Marchand et al., "High-level autoenhanced expression of a single-copy gene in Escherichia coli: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements", GENE, 262(1-2), 2001, pp. 231-238

Abstract

Bacteriophage T7 early gene 0.7 assists phage growth under suboptimal conditions ('helper' function). Whereas the C-terminal one-third of the encodedprotein participates in host transcription shutoff, the N-terminal two-thirds harbours a protein kinase ('PK') activity with broad specificity. However, how this activity relates to helper function is unclear. Here, a truncated gene 0.7 encoding PK was fused to an IPTG-inducible T7 late promoter and to a translation initiation region from a T7 late gene, and inserted intothe chromosome of an E. coli strain expressing T7 RNA polymerase. After induction, total protein synthesis remains unchanged but with over 40% devoted to PK synthesis, an amazing figure for the expression of a single-copy gene. Mutations abolishing PK activity reduce this expression by 3-fold. Thus, PK activity stimulates PK expression when the latter is controlled by T7 late genetic elements. Further experiments show that stimulation occurs at both transcriptional and post-transcriptional levels. The helper function may therefore correspond to a PK-mediated stimulation of late expression, the mechanism of which is discussed. The possibility of exploiting the PK activity fur improving E. coli expression systems is also considered. (C) 2001Elsevier Science B.V. All rights reserved.

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Documento generato il 12/07/20 alle ore 08:44:45