This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.
Bibliographical noteFunding Information:
ACKNOWLEDGMENTS This work was supported by CA96504, CA101830 and AI065824 from the National Institutes of Health, a David Koch Graduate Fellowship from the Massachusetts Institute of Technology Center for Cancer Research (G.C.), National Defense Science and Engineering Graduate Fellowships (G.C. and B.J.H.), a National Institute of General Medical Sciences Biotechnology Training Grant (S.L.S.) and a National Science Foundation Graduate Fellowship (S.M.L.). We thank the Massachusetts Institute of Technology Flow Cytometry Core Facility for their assistance, and E. Boder, J. Cochran, M. Feldhaus, M. Roguska and E. Shusta for detailed feedback on this protocol manuscript.