Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources

• Published in: Protein engineering, design and selection Vol. 26; no. 2; pp. 101 - 112
• Main Authors: Tillotson, Benjamin J., de Larrinoa, Iñigo F., Skinner, Colin A., Klavas, Derek M., Shusta, Eric V.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.02.2013
• Subjects: Antibodies; Antibody Affinity; Antibody Specificity; Antigens - immunology; Biotin; Biotinylation; Cell Extracts; Cell surface; Cell Surface Display Techniques; Detergents - chemistry; Directed Molecular Evolution; HEK293 Cells; Humans; Membrane proteins; Membrane Proteins - immunology; Octoxynol - chemistry; Original; Peptide Library; Protein Binding; Protein Engineering; Receptors, Transferrin - immunology; Saccharomyces cerevisiae; Single-Chain Antibodies - biosynthesis; Single-Chain Antibodies - genetics; Solubility; Solubilization; detergent; membrane protein; antibody; yeast surface display; affinity maturation
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzs077

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.