Zhao Qi, Zhu Zhongyu, Dimitrov Dimiter S
Protein Interactions Group, Center for Cancer Research Nanobiology Program, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
Methods Mol Biol. 2012;899:73-84. doi: 10.1007/978-1-61779-921-1_5.
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.
酵母展示是一种用于筛选具有高亲和力和热稳定性的抗体及其他蛋白质的有效技术。在此，我们描述了一种利用酵母展示对工程化抗体结构域（eAds）进行亲和力成熟优化的方法。构建了相对较大规模（约10⁹）的eAds酵母文库，并对其进行交替轮次的磁珠激活细胞分选（MACS）、荧光激活细胞分选（FACS）和随机诱变。鉴定并分析了成熟最后一轮中亲和力最高的克隆。我们对每个关键步骤进行了详细讨论，并提供了详细的实验方案和实用提示。
