title: Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.
abstract: Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.
authors.affiliation: Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Vorawit
authors.initials: V
authors.lastname: Ananphongmanee
authors.affiliation: Shrimp-Virus Interaction Laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Bangkok, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Jiraporn
authors.initials: J
authors.lastname: Srisala
authors.affiliation: Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand; Shrimp-Virus Interaction Laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Bangkok, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Kallaya
authors.initials: K
authors.lastname: Sritunyalucksana
authors.affiliation: Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Chuenchit
authors.initials: C
authors.lastname: Boonchird
mesh_terms: D000820:Animal Diseases / Q000235:genetics / Q000517:prevention & control / Q000821:virology; D000818:Animals; D061505:Cell Surface Display Techniques; D015870:Gene Expression; D005818:Genetic Engineering; D033561:Penaeidae / Q000821:virology*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D046848:White spot syndrome virus 1 / Q000235:genetics* / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: C548473:VP28 protein, white spot syndrome virus; D014759:Viral Envelope Proteins; D000090802:rab7 GTP-Binding Proteins; D020691:rab GTP-Binding Proteins