文献类型：期刊文献

中文题名：EC-SOD包涵体的柱上复性、纯化及稳定性研究

英文题名：On-column Refolding of EC-SOD Inclusion Bodies, Purification and Stability Study

作者：朱希强[1];袁勤生[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2005

卷号：32

期号：4

起止页码：101

中文期刊名：微生物学通报

外文期刊名：Microbiology China

收录：CSTPCD;;北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：重组人EC-SOD;包涵体;柱上复性;纯化

外文关键词：Recombinant hEC-SOD, Inclusion bodies, On-column refolding, Purification

摘要：利用His-tag与金属离子的特异结合作用,通过金属(Ni)亲和层析对EC-SOD包涵体进行柱上复性.考察蛋白上样量、尿素脱除速率和复性温度对复性效率的影响.利用Ni-sepharose和Heparin-sepharose亲和柱对重折叠后的蛋白进行纯化,并研究复性蛋白的稳定性.结果表明,利用Ni-sepharose亲和柱可以对EC-SOD进行复性,上样量越大,尿素脱除速率越快,复性效率越低;温度升高,复性效率增加.Ni-sepharose对复性后蛋白具有纯化作用,经Heparin-sepharose亲和柱纯化后蛋白比活明显提高.复性蛋白在10℃～50℃温度范围内活性稳定,pH低于5大于10时,其稳定性显著降低,在尿素和盐酸胍溶液中复性蛋白的稳定性较弱.
EC-SOD inclusion bodies was refolded on column using metal （Ni） affinity chromatography, based on the metal-binding property of His-tag. The effect of protein amount, urea removal speed and temperature on re folding was observed. We compared the different efficiency purified with Ni-sepharose and Heprain-sepharose affinity chromatography, and studied the stability of the refolded proteins. The results indicate that the inclusion bodies can be renatured with Ni-sepharose affinity chromatography. The increase of the protein amount and urea removal rate , the lower of the renaturation efficiency. Higher temperature was benefit to protein renaturation. Both the Ni-sepharose and Heprain-sepharose affinity column can be used to purified the refolded proteins, but purified by Heprain-sephamse affinity column the protein had higher activity. The activity of renatured protein was stable in 10 ℃ - 50℃, when pH 〈 5 or pH 〉 l0 its stability was lower significantly. In denaturating solution the stability of renatured protein was low.