研究生: |
賴千豐 Chien-feng Lai |
---|---|
論文名稱: |
具streptavidin親合性標籤之glutathione-S-transferases
融合蛋白之研究 Study of glutathione-S-transferases fused with streptavidin-affinity peptides |
指導教授: |
陳秀美
Hsiu-mei Chen |
口試委員: |
朱義旭
Yi-hsu Ju 方翠筠 Tsuei-yun Fang |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2005 |
畢業學年度: | 93 |
語文別: | 中文 |
論文頁數: | 137 |
中文關鍵詞: | glutathione-S-transferase 、基因融合 、親和性胜肽 、卵白素 |
外文關鍵詞: | glutathione-S-transferase, avidin, affinity peptides, gene fusion |
相關次數: | 點閱:468 下載:0 |
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本研究主要延續先前利用壓電生物感測晶片及微陣列螢光分析系統對於streptavidin (SAv)進行隨機胜肽組合庫篩選之研究。將所篩得可與SAv結合的四個親合性胜肽,分別將之與glutathione-S-transferase (GST)的C端融合,建構出pGST/aj21、pGST/bj13、pGST/hm8以及pGST/strepII四個質體;最終,再將之轉入大腸桿菌內表現蛋白質(aJ21、bJ13、hM8及StrepII)。於SDS-PAGE研究中發現所表現aJ21的分子量小於29 kDa且與GST酵素相近,推測aJ21可能發生降解。最後經由MALDI-TOF與N端定序得知其外加序列以及部分GST酵素之C端都有被降解之可能。於活性分析中,於固定化或自由態之bJ13融合酵素其比活性都相當高;而hM8經由固定化之後,其比活性則僅次於bJ13。由SAv凝膠吸附分析得知bJ13及hM8為與SAv親和性較佳之融合酵素。綜合以上結果得知bJ13以及hM8的外加序列皆適用於當作親和固定化之親和性標籤,其中又以bJ13的外加序列最為理想其自由態比活性約為9 U/mg;而固定化酵素之比活性則為0.46 U/mg,最後測得之Kd值為2.61×10-6 M。
The previous researches employing biopanning microarrays, and piezoelectric biochips to screen streptavidin (SAv) affinity peptides from an E. coli flagellin-displayed random peptide library have yielded four potential peptides of high affinity(黃, 2002; 蔡, 2004). In this study, the peptides were each fused to the C terminus of glutathione-S-transferase (GST). By constructing four plasmids, named pGST/aj21, pGST/bj13, pGST/hm8, and pGST/strepII, four fusion proteins, aJ21, bJ13, hM8 and StrepII were respectively expressed in E. coli. The SDS-PAGE assay indicated that the molecular weight of aJ21 was smaller than 29 kDa and close to that of GST, suggesting that aJ21 may have been hydrolyzed by proteinase. The further analysis by MALDI-TOF and N-terminal sequencing revealed that both the fusion-tag peptide and a few residues at the C terminus of GST were possibly degraded. The enzymatic assay showed that bJ13 had the highest specific activity in both free and immobilized forms, and that the immobilized hM8 was slighty less active than bJ13. The adsorption study with SAv gels indicated that bJ13 and hM8 had better SAv affinities than the other proteins. It is concluded that the bJ13 and hM8 tag, peptides are suitable to be used as immobilization affinity tags, with the bJ13 one being the best one. The specific activity of the bJ13 fusion protein was about 9 U/mg in a free form and about 0.46 U/mg in an immobilized form, and its dissociation constant (Kd) to SAv gels was about 2.61×10-6 M.
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