Wang Xin Xiang, Shusta Eric V
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
J Immunol Methods. 2005 Sep;304(1-2):30-42. doi: 10.1016/j.jim.2005.05.006.
Yeast surface display has proven to be a powerful tool for the directed evolution of immunological proteins when soluble ligands are available (Cho, B.K., Kieke, M.C., Boder, E.T., Wittrup, K.D., Kranz, D.M., 1998. A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179; Boder, E.T., Midelfort, K.S., Wittrup, K.D., 2000. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. U. S. A. 97, 10701; Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M., Wittrup, K.D., 2000. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754; Esteban, O., Zhao, H., 2004. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81). This investigation extends the utility of this display platform by demonstrating its capacity for use in cell panning selections. This was accomplished by employing a model single-chain antibody (scFv)-hapten system that allowed for detailed investigation of the factors governing panning success. Yeast displaying anti-fluorescein scFv (4-4-20) exhibited specific interactions with the fluoresceinated endothelial cells and could be recovered from large backgrounds of irrelevant yeast in just three rounds. Successful selections required as few as 1700 fluorescein ligands per cell, and a three-round enrichment ratio of 10(6) was possible. These results indicate that yeast surface display is a viable option for use in cell or tissue-based selections.
当有可溶性配体时，酵母表面展示已被证明是一种用于免疫蛋白定向进化的强大工具（赵炳奎、基克、博德、威特鲁普、克兰兹，1998年。用于发现触发细胞活化配体的酵母表面展示系统。《免疫学方法杂志》220卷，第179页；博德、米德尔福特、威特鲁普，2000年。具有单价飞摩尔抗原结合亲和力的抗体片段的定向进化。《美国国家科学院院刊》97卷，第10701页；舒斯塔、霍勒、基克、克兰兹、威特鲁普，2000年。用于T细胞受体工程的稳定支架的定向进化。《自然生物技术》18卷，第754页；埃斯特班、赵，2004年。可溶性单链人II类主要组织相容性复合体分子的定向进化。《分子生物学杂志》340卷，第81页）。本研究通过证明其在细胞淘选筛选中的应用能力，扩展了该展示平台的实用性。这是通过使用模型单链抗体（scFv）-半抗原系统实现的，该系统允许详细研究影响淘选成功的因素。展示抗荧光素scFv（4-4-20）的酵母与荧光素化内皮细胞表现出特异性相互作用，并且只需三轮就可以从大量无关酵母背景中回收。成功的筛选每个细胞只需1700个荧光素配体，并且三轮富集比可达10(6)。这些结果表明，酵母表面展示是用于基于细胞或组织的筛选的可行选择。
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