[Purification and renaturation of recombinant human Cu, Zn-SOD by metal-chelating affinity chromatography].
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 5000 u/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 (19 kD) 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.
Liu
Jian-Rong
JR
College of Life Sciences, Hebei University, Baoding 071002, China. liujianrong63@yahoo.com.cn
Liu
Jian-Guo
JG
Zhao
Xiao-Yu
XY
Gu
Ya-Jun
YJ
D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics
D002614:Chelating Agents; D011994:Recombinant Proteins; D013482:Superoxide Dismutase