金属螯合亲和层析纯化和复性重组人铜锌超氧化物歧化酶

Purification and Renaturation of Recombinant Human Cu,Zn-SOD by Metal-Chelating Affinity Chromatography

将重组人铜锌超氧化物歧化酶(rhCu,ZnSOD)包含体(经纯化后纯度达80%以上)以稀释法或透析法初步复性后,分别再经金属螯合亲和层析(MCAC)纯化。结果透析复性样品和稀释复性样品经MCAC纯化后的rhSOD比活是各自上柱前样品比活的2.2倍和5.3倍,蛋白回收率分别为64%和25%,同时两者活性回收率皆大于130%。表明目标蛋白rhSOD在经过MCAC纯化的同时又获得进一步复性。SDSPAGE显示rhSOD为19kD的单一条带,纯度大于95%,比活达到5000umg左右,同时NBT生物活性染色鉴定显示出很强的超氧化物歧化酶活性。表明MCAC对于复性不完全的rhCu,ZnSOD而言是一种纯化和使其进一步复性的简便省时且有效的方法。该方法为以包含体形式表达的基因重组蛋白的纯化和复性提供了新思路。

Overexpression of recombinant Human Cu, Zn-Superoxide Dismutase （ rhCu, Zn-SOD） in E. coli results in the form of insoluble inclusion body. Purity of rhSOD inclusion body was over 80% by isolation and purification. After preliminary renaturation by conventional dilution or dialysis, enzyme preparations was respectively purified by using Copper Metals-Chelating Affinity Chromatography （Copper-MCAC）. RhSOD specific activity purified by MCAC （from the sample renatured partly by dialysis） was 2.2 times as much as that by dialysis and protein recovery was 64%. RhSOD specific activity purified by MCAC （from the sample renatured partly by dilution） was 5.3 times as much as that by dilution and protein recovery was 25% . The two rhSOD preparations purified by MCAC had specific activities about 5000u/mg and activity recoveries were all over 130% of the enzyme activities in the samples renatured partly by dilution or dialysis. The above-mentioned results indicated that Copper-MCAC resulted in a purification and further renaturation of target protein. SDS-PAGE showed that the target protein rhSOD （19kD） was purified homogeneously and NBT activity identification proved that the purified and renatured rhSOD had very strong SOD activity. In conclusion, Copper Metals-Chelaing Affinity Chromatography appears to be a simple, rapid and efficient procedure for purifying and further renaturing rhCu, Zn-SOD by dilution or dialysis. The method provided a new idea for purifying and renaturing recombinant proteins expressed in the form of inclusion body in E. coli.