title: Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions.
abstract: Yeast surface display, a well-established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein. Historically, yeast-displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N-terminus and one to the C-terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody-staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein-protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co-expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.
authors.affiliation: Dept. of Bioengineering, Schools of Engineering and Medicine, Stanford University, Stanford, California, USA.
authors.forename: Sungwon
authors.initials: S
authors.lastname: Lim
authors.affiliation: Dept. of Bioengineering, Schools of Engineering and Medicine, Stanford University, Stanford, California, USA.|Joint Initiative for Metrology in Biology, Stanford, California, USA.|Genome-scale Measurements Group, National Institute of Standards and Technology, Stanford, California, USA.
authors.forename: Jeff E
authors.initials: JE
authors.lastname: Glasgow
authors.affiliation: Dept. of Bioengineering, Schools of Engineering and Medicine, Stanford University, Stanford, California, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Filsinger Interrante
authors.affiliation: School of Medicine, Stanford University, Stanford, California, USA.
authors.forename: Erica M
authors.initials: EM
authors.lastname: Storm
authors.affiliation: Dept. of Bioengineering, Schools of Engineering and Medicine, Stanford University, Stanford, California, USA.|Dept. of Chemical Engineering, School of Engineering, Stanford University, Stanford, California, USA.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061505:Cell Surface Display Techniques / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C543385:AGA2 protein, S cerevisiae; D001426:Bacterial Proteins; D015815:Cell Adhesion Molecules; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Binding assay; Bioconjugation; Fluorescent protein; Sortase; Yeast surface display