https://scholars.lib.ntu.edu.tw/handle/123456789/406736
標題: | High-level expression and purification of human £^D-crystallin in Escherichia coli | 作者: | Wen W.-S. Hsieh M.-C. Wang S.S.-S. |
關鍵字: | Cataract;His-tag;Human £^D-crystallin;Optimum expression;Purification | 公開日期: | 2011 | 卷: | 42 | 期: | 4 | 起(迄)頁: | 547-555 | 來源出版物: | Journal of the Taiwan Institute of Chemical Engineers | 摘要: | Human £^D-crystallin (H£^DC), a 173 amino acid protein, is a primary protein component of the human eye lens. It is composed of two highly homologous £]-sheet domains and is associated with the development of juvenile and adult-onset cataracts. In the present study, we describe the expression, purification, and characterization of H£^DC. We optimized recombinant protein expression in an Escherichia coli expression system by investigating factors such as the type of promoter, E. coli strain, culture temperature, isopropyl £]-d-thiolgalactorpyranoside (IPTG) concentration, optical density of induction (OD600nm), and duration of IPTG induction. We then successfully purified recombinant H£^DC coupled to a six-histidine tag (6¡ÑHis-H£^DC). Our results revealed that the optimal system for 6¡ÑHis-H£^DC protein expression was culture of E. coli strain BL21(DE3) bearing the pEHisH£^DC plasmid at 30¢XC and induction with 0.5mM IPTG once the culture reached an optical density of 2.5 for a period of 8h. Circular dichroism spectroscopy and fluorescence spectroscopy demonstrated that the structural integrity of the purified 6¡ÑHis-H£^DC protein was identical to that of its native counterpart. Our further investigation also revealed that almost no structural difference was detected between H£^DC with and without His-tags. The expression and purification procedure was optimized and the resultant final yield (?23.3mg/100mL of culture medium; i.e., ?23.3mg protein produced in 100mL of culture medium) was significantly (?5-10-fold) higher than those from previous reports. ? 2010 Taiwan Institute of Chemical Engineers. |
URI: | https://scholars.lib.ntu.edu.tw/handle/123456789/406736 | ISSN: | 18761070 | DOI: | 10.1016/j.jtice.2010.10.002 |
顯示於: | 化學工程學系 |
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