title: Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae.
abstract: Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three- to tenfold improvements over wild-type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFalpha1pp) is screened for the enhanced secretion of a single-chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16-fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180-fold improvement over previous reports in the secretion of full-length, functional, glycosylated human IgG(1). The production of full-length IgG(1) at milligram per liter titers in a simple, laboratory-scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: J Andy
authors.initials: JA
authors.lastname: Rakestraw
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Sazinsky
authors.forename: Andrea
authors.initials: A
authors.lastname: Piatesi
authors.forename: Eugene
authors.initials: E
authors.lastname: Antipov
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D019020:Directed Molecular Evolution; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000906:Antibodies; C048632:MF(ALPHA)1 protein, S cerevisiae; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins