title: Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.
abstract: The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture.
authors.affiliation: Department of Chemical Engineering, University of Illinois, Urbana 61801, USA.
authors.forename: R N
authors.initials: RN
authors.lastname: Parekh
authors.forename: K D
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000096:biosynthesis* / Q000378:metabolism*; D002417:Cattle; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000502:physiology*
chemical_list: D012333:RNA, Messenger; D007611:Aprotinin