透析法和稀释法复性重组人Cu,Zn-SOD包含体

以稀释法和透析法对在E.coli中以包含体形式表达的重组人超氧化物歧化酶(rhSOD)进行复性.以8 mol/L尿素溶解分离纯化的包含体(纯度达80%以上),对其稀释或透析至尿素终浓度为1.0 mol/L后,在稀释样品中补加终浓度50μmoL/L的Cu2+和5.0μmoL/L的Zn2+,在透析样品中补加终浓度5.0μmoL/L的Cu2+和0.5μmoL/L的Zn2+,分别获得了比活3 300 u/mg和4 300 u/mg的复性rhSOD.该复性样品经铜螯合亲和层析柱纯化可获得更高比活性的rhSOD.

Renaturation of Recombinant Human Cu,Zn-Superoxide Dismutase by Dilution and Dialysis

The recombinant human Cu,Zn-superoxide dismutase(rhCu,Zn-SOD) expressed in E.coli in the form of inclusion body was renatured by dilution and dialysis.After separated and purified inclusion body(over 80% purity) was redissolved in 8 mol/L urea and centrifuged,it was diluted or dialyzed into (1.0 mol/L) urea.The diluted sample was supplemented with 50 μmoL/L Cu~(2+) and 5.0 μmoL/L Zn~(2+) and the dialyzed sample was supplemented with 5.0 μmoL/L Cu~(2+) and 0.5 μmoL/L Zn~(2+).The specific activity of the renatured rhSOD was 3 300 u/mg and 4 300 u/mg respectively.The renatured samples can be further purified by metals-chelaing affinity chromatography(MCAC).