title: Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display.
abstract: Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expected from expression levels and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, 19104, USA.
authors.forename: Ranganath
authors.initials: R
authors.lastname: Parthasarathy
authors.forename: Shyamsundar
authors.initials: S
authors.lastname: Subramanian
authors.forename: Eric T
authors.initials: ET
authors.lastname: Boder
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Discher
mesh_terms: D019943:Amino Acid Substitution; D051928:CD47 Antigen / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D051928:CD47 Antigen; C497894:CD47 protein, human; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins