Open AccessOn‐column Refolding of an Insoluble His 6 ‐tagged Recombinant EC‐SOD Overexpressed in Escherichia coli
Author(s) -
ZHU XiQiang,
LI SuXia,
HE HuaJun,
YUAN QinSheng
Publication year - 2005
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2005.00035.x
Subject(s) - chemistry , escherichia coli , recombinant dna , dimer , glutathione , affinity chromatography , inclusion bodies , urea , molecular mass , complementary dna , chromatography , enzyme , biochemistry , microbiology and biotechnology , gel electrophoresis , biology , organic chemistry , gene
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. Edited by: Chang‐De LU