Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen
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Medientyp:
E-Artikel
Titel:
Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen
Beschreibung:
<jats:title>Abstract</jats:title>
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<jats:title>Background</jats:title>
<jats:p>Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast <jats:italic>Pichia pastoris</jats:italic> GS115 by inserting the <jats:italic>HBsAg</jats:italic> gene into the alcohol oxidase 1 locus.</jats:p>
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<jats:title>Results</jats:title>
<jats:p>Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L<jats:sup>-1</jats:sup>. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the <jats:italic>on-line</jats:italic> signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth.</jats:p>
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<jats:title>Conclusion</jats:title>
<jats:p>In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.</jats:p>
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