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/10033/65055
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 Kumar; 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.
Affiliation:
Helmholtz Centre for Infection Research, Braunschweig, Germany. ursula.rinas@helmholtz-hzi.de.
Citation:
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. 2009, 8:13 Microb. Cell Fact.
Journal:
Microbial cell factories
Issue Date:
2009
URI:
http://hdl.handle.net/10033/65055
DOI:
10.1186/1475-2859-8-13
PubMed ID:
19208244
Additional Links:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=19208244
Type:
Article
Language:
en
ISSN:
1475-2859
Appears in Collections:
Publications from Division of Molekulare Struktur Biologie (MOSB)

Full metadata record

DC FieldValue Language
dc.contributor.authorGurramkonda, Chandrasekhar-
dc.contributor.authorAdnan, Ahmad-
dc.contributor.authorGäbel, Thomas-
dc.contributor.authorLünsdorf, Heinrich-
dc.contributor.authorRoss, Anton-
dc.contributor.authorNemani, Satish Kumar-
dc.contributor.authorSwaminathan, Sathyamangalam-
dc.contributor.authorKhanna, Navin-
dc.contributor.authorRinas, Ursula-
dc.date.accessioned2009-04-16T10:59:53Z-
dc.date.available2009-04-16T10:59:53Z-
dc.date.issued2009-
dc.identifier.citationSimple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen. 2009, 8:13 Microb. Cell Fact.en
dc.identifier.issn1475-2859-
dc.identifier.pmid19208244-
dc.identifier.doi10.1186/1475-2859-8-13-
dc.identifier.urihttp://hdl.handle.net/10033/65055-
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.language.isoenen
dc.relation.urlhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=19208244en
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 antigen.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for Infection Research, Braunschweig, Germany. ursula.rinas@helmholtz-hzi.de.en
dc.identifier.journalMicrobial cell factoriesen

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