Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3663
標題: 聚乙烯醇固定化金屬親和吸附材於蛋白質純化之應用
Polyvinyl alcohol-based immobilized metal affinity adsorbents for protein purification
作者: 陳怡君
Chen, Yi-Chun
關鍵字: Immobilized Metal Ion Affinity Chromatography
固定化金屬親和層析法
Polyvinyl alcohol
聚乙烯醇
出版社: 化學工程學系所
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摘要: 本研究利用聚乙烯醇 (polyvinyl alcohol,PVA)在酸性下與戊二醛 (glutaraldehyde,GA) 進行化學修改,產生類似交聯 (crosslinking) 以及接枝式 (graft) 結構的水膠 (hydrogel) 型高分子粉末作為固態載體。利用化學合成的方式接上亞胺基二乙酸 (IDA) 螯合劑,作為固定化金屬親和吸附材,螯合銅離子並應用於純化Racemase與 Epimerase的粗酵素液。文中探討各種影響蛋白質吸附量與吸附因素,例如:交聯比例、金屬離子、pH值、氯化鈉濃度等。經實驗結果,獲得最適化吸附基材交聯比例為5 %聚乙烯醇、13 mM戊二醛,交聯4小時後含水率約為78 %。螯合銅離子最適化吸附與清洗條件為37 ℃、0.05 M Tris-HCl、300 mM NaCl、pH 8,脫附條件為25 ℃、0.05 M Tris-HCl、300 mM NaCl、300 mM imidazole、pH 8,總蛋白質脫附量為52 μg / g-cPVA gel。經上述結果,cPVA gel作為固定化金屬親和吸附材應用於基因重組聚組織胺標的蛋白質的吸附,多數被吸附於基材上,可選擇不同孔洞性質的膠體,降低吸附基材上的非特定蛋白質吸附,以達到純化蛋白質之目的。
In this study, crosslinking structure or graft structure beads were prepared by a crosslink polymerization technique and modified by IDA (iminodiacetic acid), loading with Cu2+, and used as adsorbent to purify a target protein (Racemase and Epimerase) from the large volumes of liquid culture medium. In the text, the such are factors that influence the adsorption of target proteins are discussed, for example: crosslinking ratio, category of metal ion, pH ionic, salt composition and NaCl concentration in the buffer. The results indicate that the optimum adsorbent crosslinking ratio is 5 % PVA and 13 mM GA crosslinked 4 hour, and water content is 78 %. The optimum purification procedure condition is 37 ℃、300 mM NaCl、pH 8、0.05 M Tris-HCl buffer for loading, and 25 ℃、300 mM NaCl、300 mM imidazole、pH 8、0.05 M Tris-HCl buffer for elution, which results in a total protein elution for 52 μg / g-cPVA gel.
URI: http://hdl.handle.net/11455/3663
其他識別: U0005-0307200815574300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0307200815574300
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