文献类型：期刊文献

英文题名：High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells

作者：He, HJ; Yuan, QS; Yang, GZ; Wu, XF

机构：[1]Chinese Acad Sci, Shanghai Inst Biochem, Shanghai 200031, Peoples R China;[2]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[3]E China Univ Sci & Technol, Inst Biochem, Shanghai 200237, Peoples R China

年份：2002

卷号：24

期号：1

起止页码：13

外文期刊名：PROTEIN EXPRESSION AND PURIFICATION

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000173687100002)】；

语种：英文

外文关键词：EC-SOD; E. coli; baculovirus expression system; Tn-5B1-4 cell; SOD

摘要：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 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. (C) 2002 Elsevier Science (USA).