High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells.
Much is known about the physical properties of the Cu,Zn- and Mn-superoxide dismutases (SODs). However, the biochemical characteristics and pharmacological properties of extracellular (EC)-SOD have been severely limited due to difficulties in obtaining and purifying the enzyme. The EC-SOD cDNA was inserted into the Escherichia coli expression plasmid pET-28a(+) which contains the T7 promoter and transformed into the E. coli BL21(DE3). After induction with 1 mmol/L isopropyl beta-D-thiogalactoside, the recombinant human EC-SOD was highly expressed as inclusion bodies. SDS-PAGE analysis revealed that recombinant EC-SOD accumulated up to 26% of the total soluble protein of E. coli cells. The expression product was purified by a Ni(2+)-IDA-Sepharose 6B column. After the denaturing and refolding processes, the recombinant human EC-SOD retains the specific enzymatic activity of 920 U/mg of the purified product. The gene encoding human EC-SOD mature peptide was also inserted into the donor plasmid pFastBacHTb. After transposition, transfection, and amplification were performed, the recombinant baculoviruses infected the Tn-5B1-4 cells and EC-SOD was highly expressed in Tn-5B1-4 cells. SDS-PAGE and Western blot analysis revealed that the subunit molecular weight of the expression product is 28 kDa. Furthermore, recombinant human EC-SOD retains the enzymatic specific activity of 200 U/mg of the Tn-5B1-4 cell lysates.
He
Hua-Jun
HJ
State Key Laboratory of Bioreactor Engineering and Institute of Biochemistry, East China University of Science and Technology, Shanghai 200237, China.
Yuan
Qin-Sheng
QS
Yang
Guan-Zhen
GZ
Wu
Xiang-Fu
XF
D000818:Animals; D016367:Baculoviridae; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D003300:Copper / Q000737:chemistry; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies; D009036:Moths; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics*; D015032:Zinc / Q000737:chemistry
D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc