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| 題 名 | 應用親和層析技術於尿胰蛋白酶抑制劑之純化=Application of Affinity Chromatography to the Purification of Urinary Trypsin Inhibitor |
|---|---|
| 作 者 | 謝學真; 李紀人; 廖婉君; | 書刊名 | 國立臺灣大學工程學刊 |
| 卷 期 | 78 2000.02[民89.02] |
| 頁 次 | 頁33-43 |
| 分類號 | 361.4 |
| 關鍵詞 | 尿胰蛋白酶抑制劑; 親和層析; 純化; Urinary trypsin inhibitor; Affinity chromatography; Purification; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | 尿胰蛋白��抑制劑 (urinary trypsin inhibitor;簡稱UTI) 是一種多用途 藥劑,具有治療急性胰臟炎、休克、早產及血管內凝血等病症的用途。為獲得高 純度的UTI,本文介紹以親和層析法純化UTI的技術。親和層析乃利用固定在擔 體上的配體 (本研究採用胰蛋白��,即trypsin) 與UTI形成專一的結合,未結合 的雜質分子予以洗除,專一性結合的UTI分子則以溶離收集。一般而言,親和 層析之純化效果較離子交換、斥濾層析等方法為佳。本研究以 CNBr-activated-Sepharose 4B為擔體,將trypsin固定於其上形成親和吸附劑,作 為純化UTI之用。粗純的UTI原料,已達50% 左右的純度,經親和層析後最高 可得到85% 以上的純度,純化倍率為1.7倍,回收率則在60% 以上。本實驗亦 探討trypsin之固定效果,UTI與trypsin之吸附等溫曲線,以及層析流量改變之 影響。根據所得結果,建議採用的操作條件為:吸附液 (0.1M Tris-HCl with 0.1M NaCl,pH 8.0)、洗滌液 (與吸附液同)、溶離液 (含鹽類至少30mM之HCl溶液, pH 2.1),並以20 ~ 40ml/hr之流量進行親和層析純化UTI。本研究亦發現以trypsin 為配體來純化UTI會伴隨有降解物產生,為克服此項缺點,未來可考慮發展以 抗體為配體來純化UTI之親和層析技術。 |
| 英文摘要 | Urinary trypsin inhibitor (UTI) has great potential in the therapy of acute pancreatis, shock, premature delivery and intravascular coagulation. This article reports the purification of UTI using affinity chromatography. The principle of affinity chromatography is based on the specific binding between a ligand immobilized on a solid carrier and a complementary biomolecule in the sample to form a complex. After that, biomolecules that are not specifically bound are washed off. The specifically bound molecules are eluted and recovered as final products. In general, affinity chromatography is an effective method for protein purification, usually more efficient than other conventional techniques such as ion exchange and size exclusion chromatography. In this study, trypsin was chosen as a ligand and immobilized on the carrier Sepharose-4B to form the affinity adsorbent for UTI purification. The purity of UTI was increased from 50% to 85% (1.7-fold increase) by the affinity chromatography. The recovery of UTI was over 60%. This study also investigated key factors influencing the purification, including the effectiveness of the immobilization of trypsin, the adsorption curve of UTI, and the flow rate through the chromatography column. Based on the results, following operation conditions were recommended: adsorption and washing buffer (0.1M Tris-HCI with 0.1M NaCl, pH 8.0), elution buffer (HCl solution containing at least 30mM of salts, pH 2.1), flow rate (20 ~ 40ml/hr). However, the present method also resulted in the degradation of UTI, possibly caused by the use of trypsin as a ligand. This difficulty may be overcome by using antibody instead as a ligand. |
本系統中英文摘要資訊取自各篇刊載內容。