http://www.abbs.info E-mail: [email protected]
ISSN
1672-9145
Acta Biochim Biophys Sin 2005,
37(4): 265–269
CN 31-1940/Q
On-column Refolding of an Insoluble His6-tagged
Recombinant EC-SOD Overexpressed in Escherichia coli
Xi-Qiang ZHU, Su-Xia LI, Hua-Jun HE, and Qin-Sheng YUAN*
State Key Laboratory of Bioreactor Engineering and Institute of
Biochemistry, East China University of Science and Technology, Shanghai 200237,
China
Abstract 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.
Key words refolding; His6 tag; recombinant EC-SOD; inclusion body; protein subunit structure
-----------------
Received: November 29, 2004 Accepted: February 18,
2005
*Corresponding author: Tel, 86-21-64252255; Fax, 86-21-64252255; E-mail, [email protected]