Title: 開發新穎專一性泛素結合分子應用於懸浮式微陣列分析平台檢測
Development of Novel Ubiquitin Linkage-Specific Affinity Binders for Suspension Microarray Detection
Authors: 許馨云
Keywords: 泛素鍵結形式;泛素結合區位;懸浮式微陣列;ubiquitin linkage;ubiquiting binding domains(UBDs);suspension microarray
Issue Date: 2012
Abstract: 泛素(ubiquitin, Ub)在蛋白質轉譯後的修飾作用中扮演重要的角色,透過此修飾作用, 可控制細胞中關鍵訊號蛋白分子和調節蛋白質濃度,同時這種修飾作用在生物體中還 可用來調節許多不同的生物途徑以維持正常生理功能的運作。泛素的胺基酸序列中有7 個賴氨酸(Lysine, K),分別在第6,11,27,29,33,48 與63 的位置 (K6, K11, K27, K29, K33, K48, and K63)。藉由其自身在此7 個位置不同的泛素化鍵結形式與修飾程度,可 能造成不同的蛋白質與蛋白質間交互作用而引發或活化不同的下游反應。此外,越來 越多的泛素親和分子(ubiquitin binding domains, UBDs)在不同的實驗研究中被發現,是 故此計畫期望延續先前研究,探討藉由這些具特定專一親和性的胜肽作為泛素相關蛋 白質體研究的分析檢測工具之可行性。 本計畫將從已建構之對特定泛素鍵結形式具親和性的蛋白質結合區位為主要材料,進 行進一步的分子設計,評估動力學參數以優化並提高其結合親和力;另一方面,將利 用噬菌體展示技術以快速篩選出對特定泛素鍵結形式具高度專一親和性之新穎泛素結 合分子。計畫最終期望利用這些經設計與篩選後的泛素高親和分子作為懸浮式微陣列 的檢測探針,發展一多參數高通量的泛素化蛋白質分子分析平台,使這些經泛素修飾 的蛋白質分子根據不同鍵結形式而能夠分別的平行分析並研究不同鍵結可能對應的相 關功能。 在此為期三年的計畫中,待完成的工作分列如下: 第一年:針對已知之特定泛素鍵結親和性分子進行分子設計: 量測辨識分子間的動力學參數與實驗條件之最佳化 第二年:開發新穎泛素親和分子應用於辨識特殊泛素鍵結形式 (K6, K11, K27, K29 and K33): 建立快速噬菌體展示篩選流程 量測辨識分子間的動力學參數與實驗條件之最佳化 第三年:以懸浮式微陣列建構泛素化修飾之感測平台: 利用設計與篩選後之泛素高親和分子作為懸浮式微陣列的檢測探針, 設計微陣列檢測流程,建立檢測應用工具
Ubiquitin (Ub) is one of the critical player in post-translation modification (PTM) reactions which regulates various cell signaling pathways by covalently conjugation to targets. There are seven lysine residues in the Ub sequence (Lysine (K) 6, K11, K27, K29, K33, K48, and K63), the diverse linkage types can be formed by the ubiquitination of itself, thereby creating poly-ubiquitin chains and different types of linkage modification have been found to lead to various cell fates. As there are increasing ubiquitin binding domains (UBDs) identified from diverse protein sources, we proposed that these small peptide molecules may serve as useful affinity binders suitable for varied ubiquitome detection and characterization. In this proposal, we will continue the previous studies and use the already constructed Ub-linkage specific UBDs as the initial materials for optimization of the affinity between the linkage and the corresponding binders; in addition, develop a modified strategy by combining the phage display technology with the flow cytometric sorter system to screen and select the potential affinity binders for novel Ub linkages, to enable for ubiquitination profiling and other enrichment applications. The proposal will be divided into three major parts for a 3-year project, which aims to: (1) Molecular engineering of the known linkage-specific UBDs: optimize the assembly and recognition of linkage-specific polyubiquitin conjugates by UBDs trimming for known linkage-specific UBDs; (2) Development of novel linkages binders by high throughput phage display screening: develop the novel linkage specific binders by applying phage display technology for newly identified K6, K11, K27, K29 and K33 linkages; (3) Development of ubiquitination sensors based on suspension microarray: development of ubiquitin-associated PTM multiplexed assays by using the engineered and selected UBD binders as detection probes for the suspension microarray.
Gov't Doc #: NSC101-2113-M009-006-MY2
URI: http://hdl.handle.net/11536/98798
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