Yeast display of engineered antibody domains.
Yeast display is an efficient technology for selection of antibodies and other proteins with high affinity and thermal stability. Here, we describe a method for affinity maturation of engineered antibody domains (eAds) using yeast display. EAd yeast libraries of relatively large size (∼10⁹) were generated and subjected to alternating rounds of magnetic-activated cell sorting (MACS), fluorescent-activated cell sorting (FACS), and random mutagenesis. The highest affinity clones from the final round of maturation were identified and analyzed. We discuss extensively each key step, and provide detailed protocols and helpful notes.
Protein Interactions Group, Center for Cancer Research Nanobiology Program, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
Qi
Q
Zhao
Zhongyu
Z
Zhu
Dimiter S
DS
Dimitrov
D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification*; D000915:Antibody Affinity; D005434:Flow Cytometry / Q000379:methods; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D016296:Mutagenesis; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D015003:Yeasts
D000906:Antibodies; D019151:Peptide Library