Expression, high cell density culture and purification of recombinant EC-SOD in Escherichia coli.
Superoxide dismutase (SOD) catalyzes the dismutation of the biologically toxic superoxide anion into oxygen and hydrogen peroxide and is deployed by the immune system to kill invading microorganisms. Extracellular SOD (EC-SOD) is a copper- and zinc-containing glycoprotein found predominantly in the soluble extracellular compartment that consists of approximately 30-kDa subunits. Here, we purified recombinant EC-SOD3 (rEC-SOD) from Escherichia coli BL21(DE3) expressing a pET-SOD3-1 construct. Cells were cultured by high-density fed-batch fermentation to a final OD(600) of 51.8, yielding a final dry cell weight of 17.6 g/L. rEC-SOD, which was expressed as an inclusion body, comprised 48.7% of total protein. rEC-SOD was refolded by a simple dilution refolding method and purified by cation-exchange and reverse-phase chromatography. The highly purified rEC-SOD thus obtained was a mixture of monomers and dimers, both of which were active. The molecular weights of monomeric and dimeric rEC-SOD were 25,255 and 50,514 Da, respectively. The purified rEC-SOD had 4.3 EU/mg of endotoxin and the solubility of rEC-SOD was more than 80% between pH 7 and 10. In 2 L of fed-batch fermentation, 60 mg of EC-SOD (99.9% purity) could be produced and total activity was 330.24 U. The process established in this report, involving high-cell-density fermentation, simple dilution refolding, and purification with ion-exchange and reverse-phase chromatography, represents a commercially viable process for producing rEC-SOD.
Son
Young-Jin
YJ
Department of Physiology and Research Institute for Biomacromolecules, University of Ulsan College of Medicine, 388-1 PoongNap-dong, Songpa-goo, Seoul, South Korea.
Bae
Ji-Young
JY
Chong
Seon-Ha
SH
Lee
Hui Sun
HS
Mo
Sang Hyun
SH
Kim
Tae Yoon
TY
Choe
Han
H
D019149:Bioreactors / Q000382:microbiology; D002845:Chromatography; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D007218:Industrial Microbiology / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013482:Superoxide Dismutase