Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigen

2.50
Hdl Handle:
http://hdl.handle.net/10033/621054
Title:
Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigen
Authors:
Lünsdorf, Heinrich; Gurramkonda, Chandrasekhar; Adnan, Ahmad; Khanna, Navin; Rinas, Ursula
Abstract:
Abstract Background A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. Results High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. Conclusions It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.
Citation:
Microbial Cell Factories. 2011 Jun 26;10(1):48
Issue Date:
26-Jun-2011
URI:
http://dx.doi.org/10.1186/1475-2859-10-48; http://hdl.handle.net/10033/621054
Type:
Journal Article
Appears in Collections:
publications of the research group compound profiling and screening (COPS)

Full metadata record

DC FieldValue Language
dc.contributor.authorLünsdorf, Heinrichen
dc.contributor.authorGurramkonda, Chandrasekharen
dc.contributor.authorAdnan, Ahmaden
dc.contributor.authorKhanna, Navinen
dc.contributor.authorRinas, Ursulaen
dc.date.accessioned2017-08-14T10:15:19Z-
dc.date.available2017-08-14T10:15:19Z-
dc.date.issued2011-06-26en
dc.identifier.citationMicrobial Cell Factories. 2011 Jun 26;10(1):48en
dc.identifier.urihttp://dx.doi.org/10.1186/1475-2859-10-48en
dc.identifier.urihttp://hdl.handle.net/10033/621054-
dc.description.abstractAbstract Background A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. Results High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. Conclusions It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.en
dc.titleVirus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigenen
dc.typeJournal Articleen
dc.language.rfc3066enen
dc.rights.holderLünsdorf et al; licensee BioMed Central Ltd.en
dc.date.updated2015-09-04T08:22:20Zen
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