Abstract
Fluorescence-activated cell sorting (FACS) in combination with yeast surface display (YSD) has proven to be a valuable tool for the engineering of antibodies. It enables the fast and robust identification and isolation of candidates with prescribed characteristics from combinatorial libraries. A novel application for FACS and YSD that has recently evolved addresses the engineering of antibodies toward pH-switchable antigen binding, aiming at reduced binding at acidic pH, compared to neutral pH. Therefore, we give guidance for the incorporation of such pH switches into antibody variable domains using combinatorial histidine scanning libraries. The protocol describes a flow cytometric sorting technique for the enrichment of antigen-specific molecules. Moreover, we provide information on how to screen the obtained antibody pools from initial sorting to isolate and characterize pH-sensitive variants.
Similar content being viewed by others
References
Chan AC, Carter PJ (2010) Therapeutic antibodies for autoimmunity and inflammation. Nat Rev Immunol 10:301–316
Scott AM, Wolchok JD, Old LJ (2012) Antibody therapy of cancer. Nat Rev Cancer 12:278–287
Hudson PJ, Souriau C (2003) Engineered antibodies. Nat Med 9:129–134
Igawa T, Mimoto F, Hattori K (2014) pH-dependent antigen-binding antibodies as a novel therapeutic modality. Biochim Biophys Acta 1844:1943–1950
Igawa T, Ishii S, Tachibana T et al (2010) Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization. Nat Biotechnol 28:1203–1207
Murtaugh ML, Fanning SW, Sharma TM et al (2011) A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches. Protein Sci 20:1619–1631
Rhiel L, Krah S, Gunther R et al (2014) REAL-Select: full-length antibody display and library screening by surface capture on yeast cells. PLoS One 9:e114887
Boder ET, Midelfort KS, Wittrup KD (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci U S A 97:10701–10705
Tillotson BJ, de Larrinoa IF, Skinner CA et al (2013) Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources. Protein Eng Des Sel 26:101–112
Doerner A, Rhiel L, Zielonka S et al (2014) Therapeutic antibody engineering by high efficiency cell screening. FEBS Lett 588:278–287
Benatuil L, Perez JM, Belk J et al (2010) An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23:155–159
Weaver-Feldhaus JM, Lou J, Coleman JR et al (2004) Yeast mating for combinatorial Fab library generation and surface display. FEBS Lett 564:24–34
Traxlmayr MW, Lobner E, Hasenhindl C et al (2014) Construction of pH-sensitive Her2-binding IgG1-Fc by directed evolution. Biotechnol J 9:1013–1022
Schroeter C, Gunther R, Rhiel L et al (2015) A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display. MAbs 7:138–151
Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557
Rakestraw JA, Sazinsky SL, Piatesi A et al (2009) Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae. Biotechnol Bioeng 103:1192–1201
Arnold FH (1996) Directed evolution: creating biocatalystsfor the future. Chem Eng Sci 51:5091–5102
Mattanovich D, Borth N (2006) Applications of cell sorting in biotechnology. Microb Cell Fact 5:12
Gera N, Hussain M, Rao BM (2013) Protein selection using yeast surface display. Methods 60:15–26
Boder ET, Wittrup KD (1998) Optimal screening of surface-displayed polypeptide libraries. Biotechnol Prog 14:55–62
Chao G, Lau WL, Hackel BJ et al (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1:755–768
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Schröter, C. et al. (2018). Isolation of pH-Sensitive Antibody Fragments by Fluorescence-Activated Cell Sorting and Yeast Surface Display. In: Bornscheuer, U., Höhne, M. (eds) Protein Engineering. Methods in Molecular Biology, vol 1685. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7366-8_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7366-8_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7364-4
Online ISBN: 978-1-4939-7366-8
eBook Packages: Springer Protocols