title: Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources.
abstract: Antigen preparations in the form of detergent-solubilized cell lysates could, in principle, render membrane proteins (MPs) compatible with in vitro antibody engineering technologies. To this end, detergent-solubilized cell lysates were coupled with the yeast surface display platform to affinity mature an anti-transferrin receptor (TfR) single-chain antibody (scFv). Lysates were generated from TfR-expressing HEK293 cells by solubilization with detergent-containing buffer after undergoing plasma membrane-restricted biotinylation. Lysate-resident TfR was then combined with a mutagenic anti-TfR scFv library in a competitive, dissociation rate screen, and scFvs were identified with up to 4-fold improved dissociation rates on the surface of yeast. Importantly, although the lysates contained a complex mixture of biotinylated proteins, the engineered scFvs retained their TfR binding specificity. When secreted by yeast as soluble proteins, mutant scFvs bound to cell surface TfR with 3-7-fold improvements in equilibrium binding affinity. Although a known MP antigen was targeted for purposes of this study, employing biotin tagging as a means of antigen detection makes the lysate-based approach particularly flexible. We have previously shown that yeast display can be used to identify lead antibodies using cell lysate-resident MP antigens, and combined with this work showing that antibodies can also be quantitatively engineered using cell lysates, these approaches may provide a high-throughput platform for generation and optimization of antibodies against MPs.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Dr., Madison, WI 53706, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Tillotson
authors.forename: Iñigo F
authors.initials: IF
authors.lastname: de Larrinoa
authors.forename: Colin A
authors.initials: CA
authors.lastname: Skinner
authors.forename: Derek M
authors.initials: DM
authors.lastname: Klavas
authors.forename: Eric V
authors.initials: EV
authors.lastname: Shusta
mesh_terms: D000915:Antibody Affinity; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology*; D002457:Cell Extracts; D061505:Cell Surface Display Techniques; D003902:Detergents / Q000737:chemistry; D019020:Directed Molecular Evolution; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology; D017830:Octoxynol / Q000737:chemistry; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D011990:Receptors, Transferrin / Q000276:immunology*; D012441:Saccharomyces cerevisiae; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility
chemical_list: D000941:Antigens; D002457:Cell Extracts; D003902:Detergents; D008565:Membrane Proteins; D019151:Peptide Library; D011990:Receptors, Transferrin; D057127:Single-Chain Antibodies; D017830:Octoxynol