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    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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    Authors
    Gurramkonda, Chandrasekhar
    Adnan, Ahmad
    Gäbel, Thomas
    Lünsdorf, Heinrich
    Ross, Anton
    Nemani, Satish K
    Swaminathan, Sathyamangalam
    Khanna, Navin
    Rinas, Ursula
    Issue Date
    2009-02-10
    
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    Abstract
    Abstract Background 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 Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results 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-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line 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. Conclusion 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.
    Citation
    Microbial Cell Factories. 2009 Feb 10;8(1):13
    URI
    http://dx.doi.org/10.1186/1475-2859-8-13
    http://hdl.handle.net/10033/620790
    Type
    Journal Article
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    publications of the research group recombinant protein expression (RPEX)
    publications of the research group recombinant protein expression (RPEX)

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