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

Lünsdorf H, Gurramkonda C, Adnan A, Khanna N, Rinas U - Microb. Cell Fact. (2011)

Bottom Line: We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Helmholtz Centre for Infection Research (VAM), Braunschweig, Germany.

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.

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Images of recombinant P. pastoris producing HBsAg during growth on methanol. (A-D) Representative transmission electron micrographs of ultrathin sectioned cells of P. pastoris GS115 grown for 151 h on methanol as described in the Materials and Methods section. Abbreviations as specified in Figure 1.
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Figure 2: Images of recombinant P. pastoris producing HBsAg during growth on methanol. (A-D) Representative transmission electron micrographs of ultrathin sectioned cells of P. pastoris GS115 grown for 151 h on methanol as described in the Materials and Methods section. Abbreviations as specified in Figure 1.

Mentions: To determine the location and appearance of the Hepatitis B surface antigen in overproducing cells, electron microscopic studies in combination with immunogold labeling were carried out. Cells growing on glycerol and cells after induction of HBsAg synthesis through methanol feeding were first subjected to transmission electron microscopy for ultrastructural analysis (Figure 1A and 1B, respectively). The ultrastructure of the cells changed substantially after exposure to methanol. In the cytosol of HBsAg-producing cells large irregular cloud-shaped areas of medium electron density became apparent (Figure 1B). These morphological features were absent in cells growing on glycerol (Figure 1A) and also absent in cells producing insulin precursor as secreted protein after exposure to methanol (Figure 1C). Both cells producing either HBsAg or insulin precursor on methanol contained microbodies (peroxisomes) with internal crystal-like structures (Figures 1C, Figure 2, Figure 3B). These peroxisomes are typical for methylotrophic yeast cells when growing on methanol [21,22] and mainly contain enzymes necessary for the breakdown of carbon sources such as alcohols or fatty acids (e.g. alcohol oxidases, catalases, acetyl-CoA oxidases) [23-25].


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

Lünsdorf H, Gurramkonda C, Adnan A, Khanna N, Rinas U - Microb. Cell Fact. (2011)

Images of recombinant P. pastoris producing HBsAg during growth on methanol. (A-D) Representative transmission electron micrographs of ultrathin sectioned cells of P. pastoris GS115 grown for 151 h on methanol as described in the Materials and Methods section. Abbreviations as specified in Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142206&req=5

Figure 2: Images of recombinant P. pastoris producing HBsAg during growth on methanol. (A-D) Representative transmission electron micrographs of ultrathin sectioned cells of P. pastoris GS115 grown for 151 h on methanol as described in the Materials and Methods section. Abbreviations as specified in Figure 1.
Mentions: To determine the location and appearance of the Hepatitis B surface antigen in overproducing cells, electron microscopic studies in combination with immunogold labeling were carried out. Cells growing on glycerol and cells after induction of HBsAg synthesis through methanol feeding were first subjected to transmission electron microscopy for ultrastructural analysis (Figure 1A and 1B, respectively). The ultrastructure of the cells changed substantially after exposure to methanol. In the cytosol of HBsAg-producing cells large irregular cloud-shaped areas of medium electron density became apparent (Figure 1B). These morphological features were absent in cells growing on glycerol (Figure 1A) and also absent in cells producing insulin precursor as secreted protein after exposure to methanol (Figure 1C). Both cells producing either HBsAg or insulin precursor on methanol contained microbodies (peroxisomes) with internal crystal-like structures (Figures 1C, Figure 2, Figure 3B). These peroxisomes are typical for methylotrophic yeast cells when growing on methanol [21,22] and mainly contain enzymes necessary for the breakdown of carbon sources such as alcohols or fatty acids (e.g. alcohol oxidases, catalases, acetyl-CoA oxidases) [23-25].

Bottom Line: We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Helmholtz Centre for Infection Research (VAM), Braunschweig, Germany.

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.

Show MeSH
Related in: MedlinePlus