On-column refolding of an insoluble His6-tagged recombinant EC-SOD overexpressed in Escherichia coli.
The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.
Zhu
Xi-Qiang
XQ
State Key Laboratory of Bioreactor Engineering and Institute of Biochemistry, East China University of Science and Technology, Shanghai 200237, China.
Li
Su-Xia
SX
He
Hua-Jun
HJ
Yuan
Qin-Sheng
QS
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility; D013482:Superoxide Dismutase / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D006639:Histidine; C441642:SOD3 protein, human; D013482:Superoxide Dismutase