title: Design and optimization of short DNA sequences that can be used as 5' fusion partners for high-level expression of heterologous genes in Escherichia coli.
abstract: The 5' terminal nucleotide sequence of a gene is often a bottleneck in recombinant protein production. The ifn-α2bS gene is poorly expressed in Escherichia coli unless a translocation signal sequence (pelB) is fused to the 5' end of the gene. A combined in silico and in vivo analysis reported here further indicates that the ifn-α2bS 5' coding sequence is suboptimal for efficient gene expression. ifn-α2bS therefore presents a suitable model gene for describing properties of 5' fusions promoting expression. We show that short DNA sequences corresponding to the 5' end of the highly expressed celB gene, whose protein product is cytosolic, can functionally replace pelB as a 5' fusion partner for efficient ifn-α2bS expression. celB fusions of various lengths (corresponding to a minimum of 8 codons) led to more than 7- and 60-fold stimulation of expression at the transcript and protein levels, respectively. Moreover, the presence of a celB-based fusion partner was found to moderately reduce the decay rate of the corresponding transcript. The 5' fusions thus appear to act by enhancing translation, and bound ribosomes may accordingly contribute to increased mRNA stability and reduced mRNA decay. However, other effects, such as altered protein stability, cannot be excluded. We also developed an experimental protocol that enabled us to identify improved variants of the celB fusion, and one of these (celBD11) could be used to additionally increase ifn-α2bS expression more than 4-fold at the protein level. Interestingly, celBD11 also stimulated greater protein production of three other medically important human genes than the wild-type celB fragment.
authors: Department of Biotechnology.
authors: Veronika
authors: V
authors: Kucharova
authors: Jørgen
authors: J
authors: Skancke
authors: Trygve
authors: T
authors: Brautaset
authors: Svein
authors: S
authors: Valla
mesh_terms: D024506:5' Flanking Region / Q000235:genetics*; D019677:Artificial Gene Fusion / Q000379:methods*; D001483:Base Sequence; D015153:Blotting, Western; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016898:Interferon-alpha / Q000235:genetics*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D016898:Interferon-alpha; D009841:Oligonucleotides
references: J Bacteriol. 1999 Jun;181(11):3587-90
references: 10348874
references: RNA. 1998 Mar;4(3):319-30
references: 9510333
references: Nat Methods. 2011 Sep 29;8(10):785-6
references: 21959131
references: PLoS One. 2013 Jun 19;8(6):e66429
references: 23840466
references: Proc Natl Acad Sci U S A. 1990 Oct;87(19):7668-72
references: 2217199
references: Cell. 2007 Sep 21;130(6):1019-31
references: 17889647
references: Nat Biotechnol. 2009 Oct;27(10):946-50
references: 19801975
references: J Bacteriol. 2005 Feb;187(4):1344-9
references: 15687198
references: Plasmid. 1997;38(1):35-51
references: 9281494
references: Appl Environ Microbiol. 2007 Feb;73(3):906-12
references: 17142370
references: Metab Eng. 2000 Apr;2(2):79-91
references: 10935724
references: Microbiology (Reading). 1994 May;140 ( Pt 5):1183-8
references: 8025683
references: Biochem Biophys Res Commun. 2008 Feb 1;366(1):135-41
references: 18053805
references: J Bacteriol. 2007 Jan;189(2):501-10
references: 17085569
references: Gene. 2002 Apr 17;288(1-2):1-8
references: 12034488
references: Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12142-9
references: 8901547
references: Cell Mol Life Sci. 2009 Feb;66(3):423-36
references: 19011758
references: Biochem Biophys Res Commun. 2004 Sep 24;322(3):1038-44
references: 15336569
references: RNA. 2002 Sep;8(9):1137-47
references: 12358433
references: J Ind Microbiol Biotechnol. 2012 Mar;39(3):383-99
references: 22252444
references: J Biotechnol. 2012 Apr 30;158(4):224-30
references: 21801767
references: Gene. 2005 Nov 21;361:13-37
references: 16213112
references: Genes Dev. 2000 May 1;14(9):1109-18
references: 10809669
references: Plasmid. 2006 Nov;56(3):223-7
references: 16806469
references: Gene. 1989 May 15;78(1):59-72
references: 2475391
references: J Bacteriol. 1994 Mar;176(6):1683-8
references: 7510674
references: Nucleic Acids Res. 1994 Apr 11;22(7):1287-95
references: 8165145
references: Appl Microbiol Biotechnol. 2004 Jun;64(5):625-35
references: 14966662
references: Nucleic Acids Res. 2007;35(17):5748-54
references: 17717002
references: Appl Environ Microbiol. 2009 Apr;75(7):2002-11
references: 19201973
references: Science. 2009 Apr 10;324(5924):255-8
references: 19359587
references: Nucleic Acids Res. 2011 Sep 1;39(17):7702-15
references: 21685451
references: EMBO J. 1994 Aug 15;13(16):3892-901
references: 8070416
references: Mol Microbiol. 1997 Dec;26(5):1125-35
references: 9426147
references: J Biotechnol. 2005 Jan 26;115(2):113-28
references: 15607230
references: Protein Expr Purif. 2006 Feb;45(2):374-80
references: 16125408
references: Nucleic Acids Res. 2004 Sep 30;32(17):5198-205
references: 15459289
references: Methods Mol Biol. 2008;453:3-31
references: 18712296
references: Biotechnol Bioeng. 2009 Oct 15;104(3):611-6
references: 19579224
references: Microb Biotechnol. 2009 May;2(3):379-89
references: 21261932
references: Appl Environ Microbiol. 2004 Dec;70(12):7033-9
references: 15574897
references: EMBO J. 1995 Jul 3;14(13):3252-61
references: 7542588
references: Appl Environ Microbiol. 2013 Jan;79(2):559-68
references: 23144128
references: Mol Cell. 2007 Jul 6;27(1):79-90
references: 17612492
references: Gene. 2001 Jan 24;263(1-2):273-84
references: 11223267
references: Cell. 2010 Apr 16;141(2):344-54
references: 20403328
references: Genes Dev. 1992 Jan;6(1):135-48
references: 1370426
references: Nature. 2008 Jan 17;451(7176):355-8
references: 18202662
references: FEMS Microbiol Rev. 2006 Nov;30(6):967-79
references: 16989654