title: Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress.
abstract: Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus beta-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to beta-glucosidase misfolding. By increasing expression temperature from 30 degrees C up to 40 degrees C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 degrees C. We examined the effect of temperature on beta-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble beta-glucosidase. Likewise, stress on the cell caused by beta-glucosidase expression was found to be greatly reduced at 37 degrees C compared to 30 degrees C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the beta-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.
authors.affiliation: Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, DE 19716, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Smith
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Richardson
authors.forename: Anne S
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D004721:Endoplasmic Reticulum / Q000201:enzymology; D004798:Enzymes / Q000378:metabolism*; D006358:Hot Temperature; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004798:Enzymes; D011994:Recombinant Proteins; D001617:beta-Glucosidase