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

2.50
Hdl Handle:
http://hdl.handle.net/10033/620790
Title:
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
Authors:
Gurramkonda, Chandrasekhar; Adnan, Ahmad; Gäbel, Thomas; Lünsdorf, Heinrich; Ross, Anton; Nemani, Satish K; Swaminathan, Sathyamangalam; Khanna, Navin; Rinas, Ursula
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
Issue Date:
10-Feb-2009
URI:
http://dx.doi.org/10.1186/1475-2859-8-13; http://hdl.handle.net/10033/620790
Type:
Journal Article
Appears in Collections:
publications of the research group recombinant protein expression (RPEX); publications of the research group recombinant protein expression (RPEX)

Full metadata record

DC FieldValue Language
dc.contributor.authorGurramkonda, Chandrasekharen
dc.contributor.authorAdnan, Ahmaden
dc.contributor.authorGäbel, Thomasen
dc.contributor.authorLünsdorf, Heinrichen
dc.contributor.authorRoss, Antonen
dc.contributor.authorNemani, Satish Ken
dc.contributor.authorSwaminathan, Sathyamangalamen
dc.contributor.authorKhanna, Navinen
dc.contributor.authorRinas, Ursulaen
dc.date.accessioned2017-01-27T11:53:19Z-
dc.date.available2017-01-27T11:53:19Z-
dc.date.issued2009-02-10en
dc.identifier.citationMicrobial Cell Factories. 2009 Feb 10;8(1):13en
dc.identifier.urihttp://dx.doi.org/10.1186/1475-2859-8-13en
dc.identifier.urihttp://hdl.handle.net/10033/620790-
dc.description.abstractAbstract 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.en
dc.titleSimple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigenen
dc.typeJournal Articleen
dc.language.rfc3066enen
dc.rights.holderGurramkonda et al.en
dc.date.updated2015-09-04T08:25:50Zen
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