High-Level Expression of Human Extracellular Superoxide Dismutase in Escherichia coli and Insect Cells

• Published in: Protein expression and purification Vol. 24; no. 1; pp. 13 - 17
• Main Authors: He, Hua-Jun, Yuan, Qin-Sheng, Yang, Guan-Zhen, Wu, Xiang-Fu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2002
• Subjects: Animals; Baculoviridae; baculovirus expression system; Cells, Cultured; Chromatography, Affinity; Cloning, Molecular - methods; Copper - chemistry; E. coli; EC-SOD; Escherichia coli; Gene Expression; Humans; Inclusion Bodies; Moths; SOD; Superoxide Dismutase - chemistry; Superoxide Dismutase - genetics; Tn-5B1-4 cell; Zinc - chemistry; SOD; Tn-5B1-4 cell; EC-SOD; baculovirus expression system; E. coli
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2001.1529

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 β-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 Ni2+-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.