Expression of fungal phytase on the cell surface ofSaccharomyces cerevisiae

Mo, Ae-Young; Park, Seung-Moon; Kim, Yun-Sik; Yang, Moon-Sik; Kim, Dae-Hyuk

doi:10.1007/bf02932297

Cited by 18 publications

(14 citation statements)

References 23 publications

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“…To construct a surfacedisplaying yeast vector, the ASP::ApxIIA#5 fusion fragment and the anchoring partner of AGA1-C320 were excised from the cloning vectors by digestion with BamHI/EcoRI and EcoRI/SalI respectively, and then ligated simultaneously to the BamHI/SalI sites in pYEGPD vector having the same restriction enzyme sites between the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the galactose-1-P uridyl transferase (GAL7) terminator. 23) The direction of the recombinant yeast vector was confirmed by restriction enzyme digestion and DNA sequencing. The resulting plasmid was denoted pYEGAXA5 (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…20,21) To anchor ApxIIA#5 to the yeast surface, an anchor DNA fragment containing the 3 0 half of the -agglutinin gene (AGA1-C320) encoding the C-terminal 320 amino acids was prepared by PCR using primers 5 0 -GGGAATTCGCC-AAAAGCTCTTTTATC-3 0 and 5 0 -GGTCGACTTAGAATAGCAGG-TAGGACA-3 0 , as described previously. 23) The resulting amplified PCR fragment of AGA1-C320 was cloned in EcoRI/SalI of pBluescript II SK (Stratagene, La Jolla, CA), analyzed by restriction enzyme digestion, and confirmed by DNA sequencing. To construct a surfacedisplaying yeast vector, the ASP::ApxIIA#5 fusion fragment and the anchoring partner of AGA1-C320 were excised from the cloning vectors by digestion with BamHI/EcoRI and EcoRI/SalI respectively, and then ligated simultaneously to the BamHI/SalI sites in pYEGPD vector having the same restriction enzyme sites between the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the galactose-1-P uridyl transferase (GAL7) terminator.…”

Section: Methodsmentioning

confidence: 99%

“…Compared to other proteins displayed on the yeast cell surface using the same expression cassette. 23) Western blot analysis required fewer yeast cells for CWP extraction, suggesting that the expression level of ApxIIA#5 is high enough for a good immune response.…”

Section: Microscopic Observation Of the Surface-displayed Apxiia-epitopementioning

confidence: 99%

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Surface-Displayed Expression of a Neutralizing Epitope of ApxIIA Exotoxin inSaccharomyces cerevisiaeand Oral Administration of It for Protective Immune Responses against Challenge byActinobacillus pleuropneumoniae

Kim¹,

Jung²,

Eom³

et al. 2010

Bioscience, Biotechnology, and Biochemistry

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A neutralizing epitope fragment of ApxIIA toxin (ApxIIA#5) of the Korean Actinobacillus pleuropneumoniae serotype 2 strain was expressed and immobilized on the cell surface of Saccharomyces cerevisiae for efficient vaccine development. Expression of ApxIIA#5 was confirmed by Western blot analysis using cell-wall proteins, and the surface display of ApxIIA#5 was further visualized under confocal microscopy. Quantitative ELISA revealed that the recombinant ApxIIA#5 directed to the cell surface consisted of approximately 16% cell-wall proteins, estimated to be 35 mg of ApxIIA#5 protein per liter of cultured cells. An immunoassay revealed that antigen-specific antibodies against ApxIIA#5 were present in the sera of mice fed recombinant ApxIIA#5-expressing yeast, but not in mice fed the wild-type nor the vector-only transformant. Moreover, the mice fed the recombinant epitope-expressing yeast were protected from injection of a lethal dose of A. pleuropneumoniae.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Surface-Displayed Expression of a Neutralizing Epitope of ApxIIA Exotoxin inSaccharomyces cerevisiaeand Oral Administration of It for Protective Immune Responses against Challenge byActinobacillus pleuropneumoniae

Kim¹,

Jung²,

Eom³

et al. 2010

Bioscience, Biotechnology, and Biochemistry

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“…Western blot analysis. Preparation of cell extracts (CFE) was conducted as previously described (41). Briefly, cells were grown for 3 days, harvested, washed twice in extraction buffer (50 mM Tris-HCl, 2 mM EDTA), and homogenized seven times in a vortex for 1 min each, with 1-min intervals of ice cooling.…”

Section: Vector Constructionmentioning

confidence: 99%

Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

Kim

Park

Kim

et al. 2011

Clin Vaccine Immunol

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A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.

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“…Preparation of cell-free extracts was conducted as previously described [31]. Briefly, cells were grown for three days, harvested, washed twice with an extraction buffer (50 mM Tris-HCl, 2 mM EDTA), and homogenized seven times in a vortex for 1 min each, with 1 min intervals of icecooling.…”

Section: Preparation Of Cell-free Extract and Culture Supernatantmentioning

confidence: 99%

Heterologous expression of cholera toxin B subunit in Saccharomyces cerevisiae

Lim

Jin

2008

Biotechnol Bioproc E

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The cholera toxin B subunit (CTB), which consists of five identical polypeptides and adopts a pentameric structure, has been shown to bind to the GM1-gangliosides at the cellular surface. Recombinant CTB has attracted much attention due to its non-toxicity and potential as a strong immunogenic antigen and immuno adjuvant for both system and mucosal immune responses. In this study, CTB was expressed in p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É and the resulting recombinant CTB was extensively characterized. PCR and back-transformation into bK=Åçäá confirmed the presence of the CTB gene-containing plasmid in the transformants and northern analysis showed the presence of the CTB-specific transcript. Western blot analysis of the yeast-derived protein extract showed the presence of CTB with mobility similar to purified CTB from sáÄêáç=ÅÜçäÉê~É suggesting that the expressed CTB assembled into the desired pentameric form. Quantitative ELISA revealed that the recombinant CTB comprised approximately 0.5~1.3% of the total cell-free extract. In addition, 0.5~2 mg of CTB protein per liter of cultured media was detected 1 day, at the earliest after cultivation. The GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) confirmed that the yeast-derived CTB bound specifically to the GM1-ganglioside receptor, indicating that it retained its native function and pentameric form, which is required for binding to intestinal epithelial cell membrane glycolipid receptors. In addition to the development of a yeast-derived edible vaccine against cholera, this study regarding the expression and assembly of recombinant CTB into biologically active oligomers in recombinant pK=ÅÉêÉîáëá~É enables the efficient production of a GRAS microorganism-based adjuvant, as well as the development of carriers for foreign vaccine molecules. © KSBB hÉóïçêÇëW=Vibrio choleraeI=ÅÜçäÉê~=íçñáå=_=ëìÄìåáíI=djNJbifp^I=Saccharomyces cerevisiae= = = = =

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Expression of fungal phytase on the cell surface ofSaccharomyces cerevisiae

Cited by 18 publications

References 23 publications

Surface-Displayed Expression of a Neutralizing Epitope of ApxIIA Exotoxin inSaccharomyces cerevisiaeand Oral Administration of It for Protective Immune Responses against Challenge byActinobacillus pleuropneumoniae

Surface-Displayed Expression of a Neutralizing Epitope of ApxIIA Exotoxin inSaccharomyces cerevisiaeand Oral Administration of It for Protective Immune Responses against Challenge byActinobacillus pleuropneumoniae

Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

Heterologous expression of cholera toxin B subunit in Saccharomyces cerevisiae

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