Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.

2.50
Hdl Handle:
http://hdl.handle.net/10033/305192
Title:
Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.
Authors:
Gurramkonda, Chandrasekhar; Zahid, Maria; Nemani, Satish Kumar; Adnan, Ahmad; Gudi, Satheesh Kumar; Khanna, Navin; Ebensen, Thomas ( 0000-0001-8906-063X ) ; Lünsdorf, Heinrich; Guzmán, Carlos A; Rinas, Ursula
Abstract:
Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.
Affiliation:
Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; International Centre for Genetic Engineering and Biotechnology, New Delhi, India; Technology Research Centre, Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, USA. Electronic address: chskrg@umbc.edu.
Citation:
Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties. 2013, 940:104-11 J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
Journal:
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
Issue Date:
1-Dec-2013
URI:
http://hdl.handle.net/10033/305192
DOI:
10.1016/j.jchromb.2013.09.030
PubMed ID:
24141044
Type:
Article
Language:
en
ISSN:
1873-376X
Appears in Collections:
publications of the research group recombinant protein expression (RPEX)

Full metadata record

DC FieldValue Language
dc.contributor.authorGurramkonda, Chandrasekharen
dc.contributor.authorZahid, Mariaen
dc.contributor.authorNemani, Satish Kumaren
dc.contributor.authorAdnan, Ahmaden
dc.contributor.authorGudi, Satheesh Kumaren
dc.contributor.authorKhanna, Navinen
dc.contributor.authorEbensen, Thomasen
dc.contributor.authorLünsdorf, Heinrichen
dc.contributor.authorGuzmán, Carlos Aen
dc.contributor.authorRinas, Ursulaen
dc.date.accessioned2013-11-11T13:54:47Zen
dc.date.available2013-11-11T13:54:47Zen
dc.date.issued2013-12-01en
dc.identifier.citationPurification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties. 2013, 940:104-11 J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.en
dc.identifier.issn1873-376Xen
dc.identifier.pmid24141044en
dc.identifier.doi10.1016/j.jchromb.2013.09.030en
dc.identifier.urihttp://hdl.handle.net/10033/305192en
dc.description.abstractFollowing earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.en
dc.language.isoenen
dc.rightsArchived with thanks to Journal of chromatography. B, Analytical technologies in the biomedical and life sciencesen
dc.titlePurification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.en
dc.typeArticleen
dc.contributor.departmentDepartment of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; International Centre for Genetic Engineering and Biotechnology, New Delhi, India; Technology Research Centre, Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, USA. Electronic address: chskrg@umbc.edu.en
dc.identifier.journalJournal of chromatography. B, Analytical technologies in the biomedical and life sciencesen
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