High-level autoenhanced expression of a single-copy gene in Escherichia coli: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements.
Bacteriophage T7 early gene 0.7 assists phage growth under suboptimal conditions ('helper' function). Whereas the C-terminal one-third of the encoded protein 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 into the 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 for improving E. coli expression systems is also considered.
Marchand
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I
Laboratoire de Génétique Moléculaire (CNRS UMR 8541), ENS, 46 rue d'Ulm, 75230, Paris, France.
Nicholson
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AW
Dreyfus
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M
D017123:Bacteriophage T7 / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D014764:Viral Proteins; C072445:0.7 protein, Enterobacteria phage T7; D017346:Protein Serine-Threonine Kinases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases