IS2-mediated re-arrangement of the promoter sequence suppresses metabolic burden of the recombinant plasmid
Jazyk angličtina Země Spojené státy americké Médium print
Typ dokumentu srovnávací studie, časopisecké články, práce podpořená grantem
PubMed
16408844
DOI
10.1007/bf02931406
Knihovny.cz E-zdroje
- MeSH
- chloramfenikol-O-acetyltransferasa genetika metabolismus MeSH
- DNA sondy MeSH
- Escherichia coli enzymologie genetika MeSH
- genetická transkripce MeSH
- genetické vektory MeSH
- penicilinamidasa genetika metabolismus MeSH
- plazmidy * MeSH
- polymerázová řetězová reakce metody MeSH
- promotorové oblasti (genetika) * MeSH
- regulace genové exprese u bakterií * MeSH
- rekombinace genetická * MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- transpozibilní elementy DNA * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- chloramfenikol-O-acetyltransferasa MeSH
- DNA sondy MeSH
- penicilinamidasa MeSH
- rekombinantní proteiny MeSH
- transpozibilní elementy DNA * MeSH
Recombinant plasmid pKA18 of the high expression bacterial system for penicillin amidase ('penicillin G acylase') bears the 3' end region of IS2 element. The IS2 sequence replaces the -35 region of promoter of pga and extends up to TAGTAT box at position -10 of the promoter region. It therefore forms a hybrid promoter of pga ppgaHT. A natural promoter ppgaWT was not detected on any recombinant plasmid isolated from recombinant strains of Escherichia coli constitutively producing penicillin amidase. PCR fragments carrying both types of promoters were cloned into the promoter-probe vector pET2 to compare their transcriptional activity: the activity of ppgaWT was 5x higher than that of ppgaHT. The same nucleotide "G" localized 28 nucleotides upstream of the translation start point was identified as the respective transcription start point of both mRNAs. An attempt was made to place the pga gene cloned on a plasmid under the control of the natural promoter: not a single clone expressing penicillin amidase was found among 150 transformants. High transcriptional activity of the natural promoter together with high pga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme.
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