翘嘴鳜铜锌超氧化物歧化酶重组蛋白表达、纯化及特性分析

超氧化物歧化酶(SOD)是清除生物体内超氧阴离子自由基的一种重要抗氧化酶。根据翘嘴鳜(Siniperca chuatsi)Cu/Zn-SOD基因序列(Gen Bank登录号:KJ558392.1)设计表达引物,扩增获得截去信号肽后的一段460 bp的序列,序列经过鉴定后,构建了重组表达质粒p ET-30a+Sc Cu/Zn-SOD,并将该质粒转入到BL21(DE3)中,用IPTG诱导进行表达。经过优化表达条件得到可溶的Sc Cu/Zn-SOD重组蛋白(rScCu/Zn-SOD),纯化重组蛋白后测定rScCu/Zn-SOD的浓度和酶活性。结果发现在20℃和37℃条件下均能够诱导Sc Cu/ZnSOD的表达。37℃时重组蛋白主要以包涵体形式存在。降低诱导温度和补充Cu~(2+)/Zn~(2+)可提高rScCu/Zn-SOD的表达量。在20℃、0.5 mmol/L IPTG条件下,添加0.5 mmol/L CuSO_4和0.1 mmol/L ZnCl_2于培养基中,重组蛋白的表达量明显升高。纯化后的重组蛋白浓度为0.14 mg/m L,酶活力为108.5 U/mg。rScCu/Zn-SOD最适温度为37℃,最适pH为7.0,可耐受5%浓度的SDS蛋白质变性剂。

Analysis and characterization on recombinant protein of Cu/Zn superoxide dismutase from Siniperca chuatsi

Superoxide dismutases ( SODs) are one family of important antioxidant enzymes involved in scavenging superox-ide anion free radical in organisms. In the present paper, the expression primer was designed by Cu/Zn-SOD gene se-quence of Siniperca chuatsi ( named as ScCu/Zn-SOD, accession numbers: KJ558392. 1 ) . A truncated signal peptide sequence with 460 bp was amplified. After identification of the sequence, the constructed recombinant expression plasmid (pET-30a+ScCu/Zn-SOD) was transformed into BL21(DE3), and was expressed by induction with IPTG. The solu-ble recombinant protein of ScCu/Zn-SOD ( designated as rScCu/Zn-SOD) was obtained by optimized expression condi-tion, and the concentration and activity of rScCu/Zn-SOD was measured after the purification of recombinant protein. The results showed that the expression of ScCu/Zn-SOD could be induced under 20℃ and 37℃ conditions. The rScCu/Zn-SOD was mainly aggregated to form inclusion bodies at 37 ℃. The expression quantity of rScCu/Zn-SOD could be im-proved by reduction induction temperature and supplement Cu2+ /Zn2+. The addition of 0. 5 mmol/L CuSO4 and 0. 1 mmol/L ZnCl2 in culture medium was optimal under 20 ℃ and 0. 5 mmol/L IPTG condition, and the expression quantity of rSc-Cu/Zn-SOD was obviously increased. The results can provide basic data for the research on functional characterization of SOD protein.