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標題: | 抗體多變域中環狀區的序列特徵與其穩定性決定因子 Loop sequence features and stability determinants in antibody variable domains |
作者: | Hung-Ju Chang 張弘儒 |
指導教授: | 楊安綏(An-Suei Yang) |
關鍵字: | 噬菌體呈現,次世代定序,高通量功能性分析技術,單鏈多變域片段抗體, Phage display,Next generation sequencing,High throughput functional assay,Single chain variable fragments, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 儘管相關研究已證實環狀區對於蛋白質摺疊過程的重要性,但是環狀區本身序列之多變性,對於憑藉資料庫中有限結構資訊的傳統研究方式來說依舊是一大挑戰,特別是在於如何找出環狀區中對於結構穩定性有決定性影響的序列這一方面。因此,我們希望能透過結合全面性突變 (comprehensive mutagenesis)、次世代定序以及高通量功能性分析的策略來解決這個難題。由此策略所取得的大量資訊可以幫助我們有效地去進一步探討蛋白質目標區域中有關序列-結構-功能之間的關係。本研究以一個經過抗體共通性序列 (consensus sequence) 設計過的單鏈多變域片段抗體 (single chain variable fragment, scFv) 上的十數個環狀區作為我們研究的目標。與先前針對蛋白質上每一個序列位置做單點飽和突變之策略不同的是,本實驗採用同時對蛋白質上目標環狀區突變六到七個胺基酸的全面性突變方式來建構噬菌體呈現資料庫。在早期的研究當中,認為在環狀區裡重要的是其共通性序列或者是偏好形成轉折 (turn) 的胺基酸序列,但在本研究結果當中發現真正重要的應該是與形成蛋白質三級結構相關的序列。我們同時藉由電腦計算以及實驗的方式去分析各種不同的突變株群,它們其中大多數都比原生株有更好的熱穩定性與表現量。總結來說,本研究提供了一種新的策略去探索在自然界當中尚未被發現的序列與功能相關性。 Despite growing evidence indicates the importance of loop in protein folding process, the hyper variety of loop sequence composition is still the major challenge for the traditional studies which are based on limited structural information to determine the functional key residues within the loop region. This conundrum can be resolved by a new strategy based on the combination of comprehensive mutational analysis, next generation sequencing (NGS), and high throughput functional assay. The tremendous data resulting from this strategy will be able to help us exploring the sequence-structure-function relationships in local protein regions effectively. The 6 CDRs and 10 non-CDR loops in variable domains of a model single chain variable fragment (scFv) which have been optimized with consensus sequence approach are used as our study platform. Different from previous studies based on numerous sets of single site saturated mutagenesis, we have constructed the scFv synthetic libraries with 6-7 amino acids mutated within the target loop region simultaneously. The results indicate that the essential sequences within the loop regions are dictated by the residues involving tertiary interactions, rather than the consensus sequences or the sequences with high turn propensities. Groups of variants with different sequence features from the wild type sequence are characterized by computational or experimental methods, and they reveal significant improvement in thermal stability and expression level. This study elaborates a new strategy for exploring the protein fitness landscape and possible sequence-function relationship unseen in nature. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58532 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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