請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75159
標題: | 利用組合式勝?資料庫研究蛋白?之酵素特異性 Combinatorial Approach to Investigate Enzyme Specificities of Protease |
作者: | 蕭斐雅 |
出版年 : | 2000 |
學位: | 碩士 |
摘要: | 固相勝?合成法為Merrifield於1963年所提出。這項技術不但使勝?合成變得快速且簡便,更被應用到許多地方,尤其,近十五年來,固相合成法被大量的應用在合成組合式資料庫上,組合式資料庫可以從數千種合成的混合物中挑出具有生物活性的化合物,對於找尋具有生物活性之物質是一個有效的方法。本論文的目的在利用組合式勝?資料庫篩選蛋白?之酵素特異性。 我們利用固相勝?合成法合成一個內含只有P1位置作變異的勝?衍生物的資料庫,經蛋白?催化水解反應後,以高效能液相層析儀分析出水解反應最快的成分,以決定該蛋白質最適合P1位置的胺基酸殘基,之後利用“分裂-連結-混合”法,固定所找到之P1位置胺基酸殘基進一步合成改變P2位置胺酸殘基的勝?資料庫,再進行同樣的蛋白?催化水解反應,找到最適合P2位置之胺基酸殘基。 在實驗中先取幾個已知其受質專一性的蛋白?:胰蛋白?、胰凝乳蛋白?、離胺酸水解?及麩胺酸水解?進行資料庫之正確性的試驗,設明資料庫的適用性無誤後,取未知受質專一性的蛋白?TM-1篩選P1及P2位置最適合之胺基酸殘基。實驗結果顯示TM-1在P1位置偏好側鏈帶有苯環的苯丙胺酸(Phe)及酪胺酸(Tyr),以這雨個胺基酸殘基分別用“分裂-連結-混合”法的結果合成P2位置作變異的資料庫,對TM-1篩選,其實驗結果為TM-1偏好在P2位置為疏水性胺基酸殘基。因此一旦成功的建立一個合適的組合式資料庫,並找到適合的分析條件後,即可快速研究出蛋白?之酵素特異性。 The solid phase peptide synthesis method was proposed by Merrfield in 1963. The technology was not only used to make peptide synthesis faster and more simply, but also applied to many other fields. Most of all, solid phase synthesis was applied to synthesize combinatorial library recently. Combinatorial library is a powerful tool to find out active compounds from a mixture of thousands compounds. It is the efficient approach for finding the biologically active molecules. The main aim of the thesis is to establish a simple combinatorial peptide library for screening the substrate specificities of proteases. We constructed a library of tetrapeptide-ester in which all the compounds were just different in the P1 site. The protease will catalyze the hydrolysis of peptide-esters that have the suitable amino acid residue in the P1 site and release tetrapeptide from the peptide-ester library. By using high performance liquid chromatography as monitor, we could find out the peptide-ester with faster hydrolysis rate. According to the most suitable amino residue of the P1 site, the peptide-ester library in which were different in the P2 site was synthesized by the “split-couple-mix” method and repeat the protease-catalyzed hydrolysis reaction to screen the suitable amino acid residue of P2 site. Before the specificity studies of unknown protease, the accuracy of the library was tested by the proteases which their specificities were well known. The trypsin, chymotrypsin, endoproteinase Lys-C and endoproteinase Glu-C were chosen for the test, and the experimental results shown that the peptide-ester library we constructed was suitable for the enzyme specificity studies. After confirming the accuracy of the library, we used the library to study the protease TM-1 which the specificity was unknown and found that its specificity in P1 site prefers phenylalanine and tyrosine, both have phenyl group at their side chain. Based on the P1 site amino acid residue, we synthesized peptide libraries which changed the amino acid residue in the P2 site by “split-couple-mix” method and studied the P2 site of TM-1.We found that TM-I prefers hydrophobic amino acid residues at P2 site. Overall, we show that the combinatorial library approach is a simple method to investigate specificity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75159 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
沒有與此文件相關的檔案。
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。