title: Expression and purification of an immunogenic dengue virus epitope using a synthetic consensus sequence of envelope domain III and Saccharomyces cerevisiae.
abstract: A synthetic consensus gene was designed based on residues of the amino acid sequences of dengue envelope domain III (scEDIII) from all four serotypes, and codon optimization for expression was conducted using baker's yeast, Saccharomyces cerevisiae. The synthetic gene was cloned into a yeast episomal expression vector, pYEGPD-TER, which was designed to direct cloned gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator. PCR and back-transformation into Escherichia coli confirmed the presence of the scEDIII gene-containing plasmid in the transformants. Northern blot analysis showed the presence of the scEDIII-specific transcript. Western blot analysis indicated that expressed scEDIII, with mobility similar to purified EDIII from E. coli, was successfully secreted into the culture media. Quantitative ELISA revealed that the recombinant scEDIII comprised approximately 0.1-0.6% of cell-free extract. In addition, 0.1-0.6 mg of scEDIII protein per liter of culture filtrate was detected on day 1 and peaked on day 3 after cultivation. The secreted scEDIII protein can be purified to ≥90% purity with 85% recovery using a simple ion-exchange FPLC followed by molecular weight cut-off. Upon administration of the purified protein to mice, mouse sera contained antibodies that were specific to all four serotypes of dengue virus. Moreover, a balanced immune response against all four serotypes was observed, suggesting that it may be possible to develop an effective tetravalent dengue vaccine using S. cerevisiae.
authors: Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
authors: Ngoc-Luong
authors: NL
authors: Nguyen
authors: Jung-Mi
authors: JM
authors: Kim
authors: Jin-Ah
authors: JA
authors: Park
authors: Seung-Moon
authors: SM
authors: Park
authors: Yong-Suk
authors: YS
authors: Jang
authors: Moon-Sik
authors: MS
authors: Yang
authors: Dae-Hyuk
authors: DH
authors: Kim
mesh_terms: D000818:Animals; D000917:Antibody Formation; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D016384:Consensus Sequence; D053059:Dengue Vaccines / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003716:Dengue Virus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000956:Antigens, Viral; D053059:Dengue Vaccines; D000939:Epitopes; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins