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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-?2bS 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-?2bS 5' coding sequence is suboptimal for efficient gene expression. ifn-?2bS 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-?2bS 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-?2bS 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.

SUBMITTER: Kucharova V 

PROVIDER: S-EPMC3811499 | BioStudies | 2013-01-01T00:00:00Z

REPOSITORIES: biostudies

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