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Screening for APOE- or RAGE-interacting Peptides using Bacterial Display Technique
APOE,RAGE,bacterial surface display,random peptide library,flow cytometry,
|Publication Year :||2018|
|Abstract:||阿茲海默症是一種起病因尚不明確的不可逆、進行性神經系統退行性疾病，特徵主要為澱粉樣蛋白斑塊(amyloid plaques)及神經原纖維纏(neurofibrillary tangles)的形成、腦中神經細胞間連結的減少和神經細胞的凋亡。絕大多數患者的阿茲海默症屬於晚發型，發生在60歲以後，雖然晚發型阿茲海默症至今還未發現特定的致病基因，但研究表明一些危險因子(risk factor)會增加病情惡化的風險，比如E型載脂肪蛋白 (APOE， Apolipoprotein E)、晚期糖化終產物受體(RAGE，receptor of product of advanced glycation end products)。為了得到能與APOE、RAGE產生相互作用的肽 （peptide），並以此肽為基礎進行結構設計，得到可以影響APOE、RAGE作用的肽類藥物。在本論文中利用細菌表面展示技術進行篩選。在篩選之前，需先得到APOE和RAGE蛋白。首先建構包含glutathione S-transferase (GST)-及APOE或RAGE基因之質体，轉化至大腸桿菌中表現出GST-APOE和GST-RAGE融合蛋白，並試圖採用親合性和分子篩層析法純化。但由於APOE表達量受限，無法得到過表達的蛋白進行後續純化；而RAGE又因是一個跨膜蛋白，並且膜外疏水性側鏈聚集纏繞導致形成沒有活性的包涵體（inclusion body），雖嘗試藉由將包涵體變性、再以透析復性等方法處理，但在復性過程中蛋白質構型發生改變，無法得到可直接應用於篩選之純化蛋白，因此利用抗原抗體特異性作用幫助篩選進行。
為了高效率篩選互作多肽，利用細菌表面展示隨機肽庫結合流式細胞分選技術。細菌細胞表面展示技術是通過重組 DNA 技術，將外源功能蛋白表達並定位於特定細菌細胞的表面。本論文藉由在細菌表面展示隨機15肽庫，篩選出能夠與RAGE產生相互作用的肽，並經dot-blot初步鑒定結合能力，並測序分析肽序列，為下一步研究提供基礎。
Alzheimer's disease is an irreversible and progressive neurodegenerative disorder with unclear causes. It is characterized by the formation of amyloid plaques and neurofibrillary tangles, decrease of interneuronal connections and apoptosis of nerve cells in the brain. The vast majority of Alzheimer's disease belongs to late-onset and occurs after the age of 60. Although late-onset Alzheimer's disease has not yet been found to have a specific causative gene, studies have shown that some risk factors may increase the risk of disease progression, such as APOE (apolipoprotein E) and RAGE (receptor of product of advanced glycation end products). To identify peptides that bind to APOE or RAGE and design peptide drugs which can modulate the disease-associated activities of these risk factors, it is desirable to obtain pure APOE and RAGE first. Plasmid constructs encoding fusion proteins of glutathion S-transferase and APOE/RAGE were used to overexpress GST-APOE and GST-RAGE fusion protein in Escherichia coli, which were later purified using affinity chromatography and size exclusion chromatography. However, the expression level of GST-APOE was too low to be purified; RAGE, on the other hand, formed inclusion bodies, probably due to its being a transmembrane protein with hydrophobic side chains aggregated and entangled out of the membrane. Although we tried to denature/renature inclusion bodies to obtain functioning RAGE, as protein conformation seemed to be changed during the renaturation process, the attempt to obtain purified protein used directly for screening purpose was unsuccessful. GST-protein-peptide interaction was therefore monitored by indirect antigen-antibody specific interaction instead.
In order to screen for interacting peptides efficiently, a bacterial surface display random peptide library combined with flow cytometric sorting technology was applied. Bacterial cell surface display technology uses recombinant DNA technology to express and localize foreign functional proteins on the surface of specific bacterial cells. In this dissertation, a random 15 peptide library was designed to screen out RAGE-interacting peptides, whose binding ability were preliminarily verified by dot-blot analysis. The identities of the candidate RAGE-interacting peptides were determined by sequencing analysis and subject to further studies.
|Appears in Collections:||藥學系|
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