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The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5'-untranslated part of mRNA.

Berg L, Lale R, Bakke I, Burroughs N, Valla S - Microb Biotechnol (2009)

Bottom Line: All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site.For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability.The two UTR sequences also stimulated expression from a constitutive σ(70)-dependent promoter (P1/P(anti-tet)), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

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Related in: MedlinePlus

β‐Lactamase activity (grey) and bla transcript (white) amounts for pIB11 with the UTR DNA sequences LII‐7 to LII‐12 (A) and LV‐1 and LV‐2 (B) relative to the wild type, expressed in E. coli DH5α. The values are the average of at least two biological recurrences, and the error bars show the deviation between them. Wild‐type values are arbitrarily set to 1.
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f2: β‐Lactamase activity (grey) and bla transcript (white) amounts for pIB11 with the UTR DNA sequences LII‐7 to LII‐12 (A) and LV‐1 and LV‐2 (B) relative to the wild type, expressed in E. coli DH5α. The values are the average of at least two biological recurrences, and the error bars show the deviation between them. Wild‐type values are arbitrarily set to 1.

Mentions: The very strong increases in ampicillin resistances displayed by the mutants containing the LII‐1 to ‐12 sequences were assumed to be a direct result of enhancement of β‐lactamase production. An obvious explanation could be that the transcripts produced were more efficiently translated than the corresponding wild‐type transcripts. To investigate this we selected six of these mutants (carrying LII‐7 to ‐12) for further studies, and first quantified the β‐lactamase activities of the corresponding host cells. The level of stimulation under induced conditions relative to the wild‐type was as high as up to about 20‐fold (LII‐11; Fig. 2A), an unexpectedly high number taken into consideration that the wild‐type system has been shown to be capable of industrial level production of recombinant proteins (see Introduction).


The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5'-untranslated part of mRNA.

Berg L, Lale R, Bakke I, Burroughs N, Valla S - Microb Biotechnol (2009)

β‐Lactamase activity (grey) and bla transcript (white) amounts for pIB11 with the UTR DNA sequences LII‐7 to LII‐12 (A) and LV‐1 and LV‐2 (B) relative to the wild type, expressed in E. coli DH5α. The values are the average of at least two biological recurrences, and the error bars show the deviation between them. Wild‐type values are arbitrarily set to 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815758&req=5

f2: β‐Lactamase activity (grey) and bla transcript (white) amounts for pIB11 with the UTR DNA sequences LII‐7 to LII‐12 (A) and LV‐1 and LV‐2 (B) relative to the wild type, expressed in E. coli DH5α. The values are the average of at least two biological recurrences, and the error bars show the deviation between them. Wild‐type values are arbitrarily set to 1.
Mentions: The very strong increases in ampicillin resistances displayed by the mutants containing the LII‐1 to ‐12 sequences were assumed to be a direct result of enhancement of β‐lactamase production. An obvious explanation could be that the transcripts produced were more efficiently translated than the corresponding wild‐type transcripts. To investigate this we selected six of these mutants (carrying LII‐7 to ‐12) for further studies, and first quantified the β‐lactamase activities of the corresponding host cells. The level of stimulation under induced conditions relative to the wild‐type was as high as up to about 20‐fold (LII‐11; Fig. 2A), an unexpectedly high number taken into consideration that the wild‐type system has been shown to be capable of industrial level production of recombinant proteins (see Introduction).

Bottom Line: All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site.For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability.The two UTR sequences also stimulated expression from a constitutive σ(70)-dependent promoter (P1/P(anti-tet)), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

Show MeSH
Related in: MedlinePlus