title: A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping.
abstract: Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full-length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full-length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark-derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A-GFP system. Yeast library screening of full-length vNAR variants which were detected via GFP expression yielded the same high-affinity binder that had previously been isolated by our group using the conventional epitope tag-based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost-friendly alternative to conventional epitope tag detections.
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Julius
authors.initials: J
authors.lastname: Grzeschik
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Steffen C
authors.initials: SC
authors.lastname: Hinz
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Doreen
authors.initials: D
authors.lastname: Könning
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Pirzer
authors.affiliation: Protein Engineering and Antibody Technologies, Merck Serono, Merck KGaA, Darmstadt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zielonka
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology; D015202:Protein Engineering / Q000379:methods*; D012270:Ribosomes / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D000939:Epitopes; D061905:Single-Domain Antibodies; D049452:Green Fluorescent Proteins
keywords: 2A peptide; IgNAR; Ribosomal skipping; Yeast surface display; vNAR