title: Heat shock at higher cell densities improves measles hemagglutinin translocation and human GRP78/BiP secretion in Saccharomyces cerevisiae.
abstract: The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37°C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20°C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37°C and 20°C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH.
authors.affiliation: Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.Graiciuno 8, Vilnius, LT-02241, Lithuania.
authors.forename: Rūta
authors.initials: R
authors.lastname: Zinkevičiūtė
authors.affiliation: Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.Graiciuno 8, Vilnius, LT-02241, Lithuania.
authors.forename: Edita
authors.initials: E
authors.lastname: Bakūnaitė
authors.affiliation: Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.Graiciuno 8, Vilnius, LT-02241, Lithuania.
authors.forename: Evaldas
authors.initials: E
authors.lastname: Čiplys
authors.affiliation: Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.Graiciuno 8, Vilnius, LT-02241, Lithuania.
authors.forename: Raimundas
authors.initials: R
authors.lastname: Ražanskas
authors.affiliation: Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.Graiciuno 8, Vilnius, LT-02241, Lithuania.
authors.forename: Jurgita
authors.initials: J
authors.lastname: Raškevičiūtė
authors.affiliation: Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.Graiciuno 8, Vilnius, LT-02241, Lithuania. Electronic address: rimantas.slibinskas@bti.vu.lt.
authors.forename: Rimantas
authors.initials: R
authors.lastname: Slibinskas
mesh_terms: D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology*; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D006389:Hemagglutinins, Viral; D011994:Recombinant Proteins; C032397:hemagglutinin protein G, measles virus