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KMID : 1094720130180040742
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Biotechnology and Bioprocess Engineering
2013 Volume.18 No. 4 p.742 ~ p.750
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High production of recombinant Norwegian salmonid alphavirus E1 and E2 proteins in Escherichia coli by fusion to secretion signal sequences and removal of hydrophobic domains
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Tøndervik Anne
Balzer Simone
Haugen Tone
Sletta Havard
Rode Marit
Lindmo Karine
Ellingsen Trond E.
Brautaset Trygve
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Abstract
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The Norwegian salmonid alphavirus (NSAV) infects farmed Atlantic salmon and rainbow trout, causes pancreas disease and leads to economic losses and fish health issues for the aquaculture. Vaccines are available, but recurring infection outbreaks at Norwegian fish farms have led to endeavours in finding solutions for increased prevention. The NSAV E1 and E2 envelope proteins are potential targets for production of recombinant subunit vaccines and for generation of antibodies for diagnostics. Efficient expression of target proteins is necessary for these applications, and here we present a new strategy for expressing this kinds of viral proteins. We show that 5′-terminal fusion of signal sequences OmpA and CSP to the e1 and e2 genes and removal of the C-terminal hydrophobic interaction and transmembrane domains of E1 and E2 leads to significantly increased expression levels. Recombinant Escherichia coli strains for high-level production of E1 and E2 harbouring these modifications were established using the inducible XylS/Pm expression cassette. Furthermore, reduction of temperature to 16°C after induction leads to 4-fold increase in production for E1, and under high-cell-density cultivations we obtained production levels up to 2.3 g/L. We also show that these proteins can be purified from inclusion bodies by affinity chromatography. This demonstrates the present approach as promising for large scale production of such viral proteins.
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KEYWORD
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recombinant vaccines, recombinant gene expression, XylS/Pm, high-cell-density cultivations, signal sequences
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