Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions

• Published in: Biotechnology journal 2017-05, Vol.12 (5), p.n/a
• Main Authors: Lim, Sungwon, Glasgow, Jeff E., Filsinger Interrante, Maria, Storm, Erica M., Cochran, Jennifer R.
• Format: Article
• Language: English
• Subjects: Aminoacyltransferases - chemistry; Aminoacyltransferases - genetics; Aminoacyltransferases - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding assay; Bioconjugation; Cell Adhesion Molecules - chemistry; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Cell Surface Display Techniques - methods; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Enzyme Assays - methods; Fluorescent protein; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Models, Molecular; Protein Binding; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sortase; Yeast surface display
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201600696

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. Yeast surface display is a well‐established technology used to analyze and engineer proteins with increased binding affinity, thermal stability, or catalytic properties. This study describes a new yeast surface display strategy that utilizes both the N‐ and C‐termini of Aga2p to co‐express two heterologous proteins. This system was used to simplify quantification of protein–protein binding interactions and to measure enzyme expression and catalytic activity on the surface of yeast.