Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments
Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments
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DOI：
10.1016/j.jim.2004.01.024
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发表时间：
2004-04-01
影响因子：
2.2
通讯作者：
Wittrup, KD
中科院分区：
文献类型：
--
作者：
Cochran, JR;Kim, YS;Wittrup, KD
细胞外蛋白的单个结构域是生物化学表征配体/受体相互作用和抗体结合位点的潜在试剂。本文以表皮生长因子受体（EGFR）为模型系统，描述了一种鉴定和表征酿酒酵母细胞表面显示的稳定蛋白结构域的方法。EGFR片段在酵母细胞表面成功表达。用构象特异性的EGFR抗体检测，酵母显示的EGFR片段被正确折叠。热变性酵母显示的EGFR蛋白区分线性和构象抗体表位。此外，egfr特异性抗体根据其竞争配体结合的能力进行分类，这已被证明具有治疗意义。基于荧光竞争结合试验确定重叠的EGFR抗体表位。酵母表面展示是一种有用的方法，可以从多结构域细胞外受体中鉴定稳定的折叠蛋白结构域，以及鉴定抗体结合表位，而不需要可溶性蛋白的表达和纯化。(C) 2004 Elsevier B.V.版权所有
Individual domains from extracellular proteins are potential reagents for biochemical characterization of ligand/receptor interactions and antibody binding sites. Here, we describe an approach for the identification and characterization of stable protein domains with cell surface display in Saccharomyces cerevesiae, using the epidermal growth factor receptor (EGFR) as a model system. Fragments of the EGFR were successfully expressed on the yeast cell surface. The yeast-displayed EGFR fragments were properly folded, as assayed with conformationally specific EGFR antibodies. Heat denaturation of yeast-displayed EGFR proteins distinguished between linear and conformational antibody epitopes. In addition, EGFR-specific antibodies were categorized based on their ability to compete ligand binding, which has been shown to have therapeutic implications. Overlapping EGFR antibody epitopes were determined based on a fluorescent competitive binding assay. Yeast surface display is a useful method for identifying stable folded protein domains from multidomain extracellular receptors, as well as characterizing antibody binding epitopes, without the need for soluble protein expression and purification. (C) 2004 Elsevier B.V. All rights reserved.