Cloning and inducible synthesis of poliovirus non-structural proteins in Saccharomyces cerevisiae.
Several coding regions of the poliovirus (PV) genome were cloned in the yeast Saccharomyces cerevisiae, and placed under the control of the inducible hybrid promoter pGAL/CYC, such that expression was triggered by incubating the cells in galactose (Gal)-containing medium. A number of PV genes encoding non-structural proteins, including 2Apro, 2B, 2C, 3A, 3AB and 3Cpro, were cloned and expressed in this eukaryotic system. The presence of these proteins after induction was detected by immunoblot analysis using specific antisera against each protein. The levels and the kinetics of protein synthesis after induction varied according to the PV protein analyzed. Thus, 2C was detected soon after Gal addition (3-5 h) and was one of the major polypeptides synthesized by yeast cells after 16 h of induction. In contrast, only low levels of synthesis were observed for 3A or 3AB, and then only after several hours of growth in Gal. The induction of the PV protease, 2Apro, was highly toxic for the cells such that growth was arrested after 5 h of induction and cell survival sharply declined.
Barco
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Centro de Biología Molecular, Universidad Autónoma de Madrid, Spain.
Carrasco
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D000086042:3C Viral Proteases; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000502:physiology; D005690:Galactose / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D017955:Poliovirus / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D008156:Luciferases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D005690:Galactose