title: Expression and characterization of infectious bursal disease virus polyprotein in yeast.
abstract: Various expression vectors containing a cDNA fragment encoding all but the first five amino acids (aa) of the large polyprotein (N-VP2-VP4-VP3-C) of infectious bursal disease virus were transformed into yeasts. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, co- or post-translational processing of the unfused large polyprotein occurred, generating a stable C-terminal product (VP3) or correct size, but without any detectable N-terminal product (VP2). Furthermore, when the processing of the polyprotein was interrupted, because of an engineered in-frame site-specific insertion of 4 aa, even VP3 (as part of the unprocessed polyprotein) was undetected. VP2 was detected in S. cerevisiae only when fused to yeast pre-sequences at the N terminus, suggesting that in yeast, VP2 or the unprocessed polyprotein, in the absence of its native N terminus or proper protection of its N-terminal aa residues is susceptible to proteolytic degradation. The first 8 aa of a modified pre-sequence of the CUP1 gene product and the pre-pro sequence of MF alpha 1 gene product have been used for stable intra- and extra-cellular production of VP2, respectively.
authors.affiliation: CSIRO, Division of Biomolecular Engineering, Parkville Laboratory, Victoria, Australia.
authors.forename: M N
authors.initials: MN
authors.lastname: Jagadish
authors.forename: P R
authors.initials: PR
authors.lastname: Vaughan
authors.forename: R A
authors.initials: RA
authors.lastname: Irving
authors.forename: A A
authors.initials: AA
authors.lastname: Azad
authors.forename: I G
authors.initials: IG
authors.lastname: Macreadie
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D007243:Infectious bursal disease virus / Q000235:genetics*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D011506:Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins