EC-SOD包涵体的柱上复性、纯化及稳定性研究

On-column Refolding of EC-SOD Inclusion Bodies, Purification and Stability Study

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.