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The 5′ terminal nucleotide sequence of a gene is often a bottleneck in recombinant protein production. Theifn-α2bS gene is poorly expressed inEscherichia coli unless a translocation signal sequence (pelB ) is fused to the 5′ end of the gene. A combinedin silico andin vivo analysis reported here further indicates that theifn-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 expressedcelB gene, whose protein product is cytosolic, can functionally replacepelB as a 5′ fusion partner for efficientifn-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 acelB -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 thecelB fusion, and one of these (celB D11 ) could be used to additionally increaseifn-α2bS expression more than 4-fold at the protein level. Interestingly,celB D11 also stimulated greater protein production of three other medically important human genes than the wild-typecelB fragment.

applied and environmental microbiology

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