title: Exploring the 5'-UTR DNA region as a target for optimizing recombinant gene expression from the strong and inducible Pm promoter in Escherichia coli.
abstract: By using the strong and inducible Pm promoter as a model, we recently reported that the β-lactamase production (encoded by bla) can be stimulated up to 20-fold in Escherichia coli by mutating the DNA region corresponding to the 5'-untranslated region of mRNA (UTR). One striking observation was the unexpected large stimulatory effect some of these UTR variants had on the bla transcript production level. We here demonstrate that such UTR variants can also be used to improve the expression level of the alternative genes celB (encoding phosphoglucomutase) and inf-α2b (encoding human cytokine interferon α2b), which both can be expressed to high levels even with the wild-type Pm UTR DNA sequence. Our data indicated some degree of context dependency between the UTR DNA and concomitant recombinant gene sequences. By constructing and using a synthetic operon, we demonstrated that UTR variants optimized for high-level expression of probably any recombinant gene can be efficiently selected from large UTR mutant libraries. The stimulation affected both the transcript production and translational level, and such modified UTR sequences therefore clearly have a significant applied potential for improvement of recombinant gene expression processes.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.
authors.forename: Laila
authors.initials: L
authors.lastname: Berg
authors.forename: Veronika
authors.initials: V
authors.lastname: Kucharova
authors.forename: Ingrid
authors.initials: I
authors.lastname: Bakke
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
mesh_terms: D020121:5' Untranslated Regions; D004247:DNA / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009876:Operon; D010733:Phosphoglucomutase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D004274:DNA, Recombinant; C554158:IFNA2 protein, human; D016898:Interferon-alpha; D012333:RNA, Messenger; D004247:DNA; D001618:beta-Lactamases; D010733:Phosphoglucomutase