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题名	离子交换色谱复性蛋白质
作者	李明
学位类别	博士
答辩日期	2003
授予单位	中国科学院过程工程研究所
授予地点	中国科学院过程工程研究所
导师	苏志国
关键词	蛋白质折叠 色谱复性 离子交换色谱 凝胶过滤色谱 蛋白质
其他题名	Protein Refolding by Ion-Exchange Chromatography LI Ming (Chemical Technology)
学位专业	生物化工
中文摘要	该文以离子交换色谱作为复性手段,对变性的鸡卵白溶菌酶、重组超氧化物歧化酶以及重组人溶菌酶的折叠复性进行了研究,并对复性后的蛋白质混合物进行了分离,用色谱方法和光谱学方法进行了性质检测.对于三种不同的蛋白质,首先使用稀释复性方法考察了各种影响因素对蛋白质折叠复性效率的影响,获得不同蛋白质在稀释复性时的优化条件.在此基础上,研究了蛋白质的离子交换色谱复性方法.采用三种缓冲液体系:(1)变性缓冲液溶解蛋白质并向色谱柱进液,使伸展的变性蛋白质吸附到离子交换色谱介质表面;(2)用复性缓冲液向色谱柱梯度进液以取代柱中的变性缓冲液,使蛋白质在色谱柱上自发地折叠;(3)用高浓度盐离子的洗脱缓冲液把折叠后的蛋白质洗脱出来.在对鸡卵白溶菌酶的色谱复性时发现,在复性缓冲液和洗脱缓冲液中加入低浓度的变性剂,可以提高蛋白质的复性效率,蛋白质的复性收率从文献报道的不到10%提高到40%.添加的变性剂可以使折叠的蛋白质分子结构比较稳定,并能提高蛋白质的比活性.
英文摘要	An ion-exchange chromatography refolding technique was developed to improve in vitro protein refolding yield. Three proteins, native hen egg white lysozyme, recombinant iron superoxide dismutase and recombinant human lysozyme, were refolded by the new refolding process. The mixtures after refolding were purified and characterized by gel filtration and other methods. These three proteins were firstly refolded by traditional dilution process to optimize the refolding condition for every protein, which helped the selection of chromatographic refolding condition. A three-buffer ion-exchange chromatographic refolding system was used to refolding these proteins. (1) Proteins that dissolved in denature buffer containing denaturant at high concentration were loaded into an ion-exchange chromatographic column, which was equilibrated with denaturant at high concentration. The stretched polypeptides were immobilized on the surface of the medium. (2) Refolding buffer was introduced gradually into the ion-exchange column, inducing the protein refolding. (3) The refolded proteins were eluted out by elution buffer containing high concentration of salt. A better result was found during hen egg white lysozyme refolding by three-buffer chromatographic system. Addition of low concentration of denaturant on refolding and elution buffers could improve mass recovery and activity recovery, keeping the active protein structure flexible, which is required for a high specific activity of an enzyme. The activity recovery of lysozyme was 40%, which is better than 10% reported in the literature. Two-buffer ion-exchange chromatography refolding system was developed to improve refolding yield. A new buffer, composing refolding buffer and elution buffer was used. This buffer, together with the denaturant buffer, formed a two-buffer system. When the new buffer was introduced into the column in a gradient manner, a gradual decrease of denaturant concentration in combination with gradual increase of ionic concentration was set up. The denatured protein would arrange its refolding structure during they adsorbed, left and re-adsorbed on the medium. The specific activity of hen egg white lysozyme, recombinant SOD, and recombinant human lysozyme from the three-buffer chromatographic system were 20000U/mg, 804U/mg and 10800U/mg, while the specific activity of them from the two-buffer system were 30390U/mg, 1020 U/mg and 19250U/mg, respectively. The later was remarkably higher than the former. The pH gradient, which was another new manner used in chromatographic refolding system, could enhance the refolding recovery during two-buffer ion-exchange chromatography refolding process. The specific activity of hen egg white lysozyme and recombinant human lysozyme were 38545U/mg and 24400U/mg in the pH gradient process, respectively. The urea gradient and the pH gradient can be integrated into one process as a dual-gradient system. During proteins refolded induced by gradient decrease of denaturant concentration, pH was gradually changed at the same time, which could improve the refolding yield. The specific activity and activity recovery of hen egg white lysozyme from dual-gradient refolding system were 42618U/mg and 95%, respectively, while recombinant SOD got 1200U/mg specific activity and 48% activity recovery. For recombinant human lysozyme, the specific activity was 27000U/mg, and activity recovery 54% from dual-gradient refolding system, respectively. In order to separate the mixture of protein after refolding, gel filtration with low concentration urea and NaCl added into elution buffer was developed. Different refolding products, such as aggregates, correctly refolded and misfolded components were characterized by gel filtration, hydrophobic interaction chromatography and other biophysical methods.
语种	中文
公开日期	2013-09-16
页码	145
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1381]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	李明. 离子交换色谱复性蛋白质[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2003.

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