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標題: | 利用表面電漿共振生物感測儀分析槲黃素與hnRNP A1結合位置及親和力 Determine the quercetin binding sites and binding affinity of hnRNP A1 by surface plasmon resonance |
作者: | Kai-Chao Hsu 許凱超 |
指導教授: | 周綠蘋 |
共同指導教授: | 詹迺立 |
關鍵字: | 槲黃素,hnRNP A1,表面電漿共振, Quercetin,hnRNP A1,surface plasmon resonance, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 槲黃素(Quercetin)是一種普遍存在於蔬果和穀物中的類黃酮化合物,在許多研究中指出,可以誘導攝護腺癌細胞凋亡並抑制癌細胞的生長,具有選擇性的殺死癌細胞而不影響到正常細胞。本實驗室在先前的研究結果中,利用化學蛋白體學的技術找出槲黃素的標靶蛋白,發現hnRNP A1(heterogeneous nuclear ribonucleoprotein A1)與槲黃素具有結合作用,hnRNP A1具有往返於細胞核和細胞質之間的能力,能將mRNA帶出細胞核。hnRNP A1的N端有兩段能與RNA結合的區域,RRM (RNA-recognition motifs);而C端則是一個M9 domain,經由Transportin (Trn1)的結合進出細胞核的訊號序列。正常功能中hnRNP A1將mRNA運送出細胞核後,hnRNP A1會從細胞質再回到細胞核。本實驗室在先前的研究發現,當細胞處理槲黃素後,hnRNP A1會漸漸堆積在細胞質中,並且影響抗凋亡蛋白的mRNA轉譯,使細胞走向凋亡。因此本研究希望利用表面電漿共振方法(Surface plasmon resonance)找出槲黃素與hnRNP A1結構上的哪一區域的親和力較高,進而得知槲黃素與hnRNP A1的結合位置。除了全長的表現質體外,我們將hnRNP A1依結構區域劃分三個區域,建構出N-terminal、middle region、C-terminal的表現質體,利用大腸桿菌表現四個重組蛋白,經由鎳離子管柱純化出具有His-tag 的重組蛋白,並使用質譜儀確認表現的蛋白質身份。為了得到更純的蛋白質,我們接著使用膠體過濾層析純化並且分析蛋白質的原態分子量。最後使用SPR生物感測儀分析槲黃素分別與這四個重組蛋白的親和力。槲黃素與hnRNP A1 全長蛋白的親和力為8.88 μM、hnRNP A1 N端為90.34 μM、hnRNP A1中段則沒有結合的訊號、hnRNP A1 N端的親和力為1.69 μM,由此可得知槲黃素與hnRNP A1的最佳結合位置在C端區域。 Quercetin, one of the most abundant flavonoids, is ubiquitous in plants. In many studies, quercetin was shown to inhibit cells growth and induction of apoptosis in prostate cancer cells. Moreover, quercetin selectively kills prostate cancer cells, but does not affect the viability of prostate normal epithelial cells. By chemical proteomics approach, we have identified hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1) as a direct target of quercetin. Previous studies indicate hnRNP A1 has ability of shuttle from nucleus and cytoplasm, mostly, binding mRNA out of nucleus. Upon quercetin treatment, quercetin binds to hnRNPA1 and hampers its returning back to nucleus resulting in accumulation of hnRNPA1 in the cytoplasm. Thus, cells are attempting towards apoptosis by eventually transporting to stress granules in the quercetin treatment. In this study, our main purpose is to figure out which region of hnRNP A1 get higher affinity with quercetin by using surface plasmon resonance assay, and further find out the quercetin binding sites of hnRNP A1. According to the structure information of hnRNP A1, we constructed three truncated proteins of hnRNP A1: N-terminal region, middle region, and C-terminal region. We used E.coli expression system to express four recombinant proteins including full length of hnRNP A1, then purified His-tag recombinant proteins by Ni2+ affinity column. LC-MS/MS was used to further confirm the sequence identity. The recombinant proteins were further purified by gel-filtration using HiLoad Superdex 16/60 FPLC column, and the result indicated that all purified proteins are monomeric forms. Finally, we investigated the affinity between quercetin and each of those four recombinant proteins by using SPR. The affinity of quercetin and full length of hnRNP A1 is 8.88 μM, N-terminal region is 90.34 μM, the middle region shows no signal, and C-terminal region is 1.69 μM. Our result indicates that the quercetin binding site of hnRNP A1 is on the C-terminl region. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58340 |
全文授權: | 有償授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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