Discovery of high-affinity peptide binders to BLyS by phage display
Discovery of high-affinity peptide binders to BLyS by phage display
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DOI：
10.1002/jmr.722
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发表时间：
2005-01-01
影响因子：
2.7
通讯作者：
Sexton, DJ
中科院分区：
文献类型：
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作者：
Fleming, TJ;Sachdeva, M;Sexton, DJ
B淋巴细胞刺激因子（BLyS）是肿瘤坏死因子（TNF）家族成员，是B细胞应答的关键调节因子。我们采用基于噬菌体展示的方法来鉴定以高选择性和亲和力结合BLyS的肽。第一代BLyS结合肽的序列分析揭示了两个主要的肽基序，包括一个含有保守的DxLT序列。发现具有该基序的所选线性肽以1-3 μ m的K-D值结合BLyS。为了提高对BLyS的结合亲和力，将DxLT序列侧翼的共有残基接种到第二代BLyS亲和力成熟文库（BAML）中。使BAML噬菌体经受严格的结合竞争条件以选择表达BLyS的高亲和力肽配体的分离物。BAML肽序列的选择后分析导致核心十肽基序（WYDPLTKLWL）的鉴定。含有该核心基序的肽表现出低至26 nm的KD值，比第一代肽低约100倍。开发荧光各向异性测定以监测钌螯合物标记的BLyS与TACI-Fc（BLyS受体的可溶形式）之间的蛋白质-蛋白质相互作用。使用该测定，发现BAML肽破坏这种高亲和力蛋白质-蛋白质相互作用。这证明了短肽破坏高亲和力精氨酸-受体相互作用的潜力。版权所有（c）2004年约翰威利父子有限公司。
B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K-D values of 1-3 mu m. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited KD values as low as 26 nm, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions. Copyright (c) 2004 John Wiley & Sons, Ltd.