钝顶螺旋藻超氧化物歧化酶的原核表达及纯化

Prokaryotic expression and purification of superoxide dismutase of Spirulina platensis

目的原核表达并纯化钝顶螺旋藻超氧化物歧化酶(superoxide dismutase,SOD)。方法利用PCR技术扩增钝顶螺旋藻sod基因,克隆入原核表达载体pGEX-4T-1,构建重组表达质粒pGEX-4T-sod,转化大肠埃希菌BL21(DE3)pLysS,IPTG诱导表达。表达的重组融合蛋白SOD-GST利用蛋白纯化树脂Glutathione SepharoseTM4 Fast Flow纯化后,采用改进的邻苯三酚自氧化法测定重组SOD的活性。结果重组表达质粒pGEX-4T-sod经双酶切和测序证实构建正确;表达的融合蛋白SOD-GST的相对分子质量约为49 000,表达量占菌体总蛋白的30.2%,在重组菌裂解上清和沉淀中均有融合蛋白的表达;纯化的融合蛋白纯度为82.4%,浓度为2.8 mg/ml,从可溶性表达产物中纯化的酶的比活性为1 440 U/mg。结论在大肠埃希菌中成功表达并纯化了具有生物学活性的钝顶螺旋藻SOD,为其产业化生产奠定了基础。

Objective To express the superoxide dismutase （SOD） gene of Spirulina platensis and purify the expressed product. Methods The sod gene of S. platensis was amplified by PCR and cloned into prokaryofic expression vector pGEX-4T-sod. The constructed recombinant plasmid pGEX-4T-sod was transformed to E. coli BL21 （DE3） pLysS for ex- pression under induction of IPTG. The expressed fusion protein SOD-GST was purified with Glutathione SepharoseTM 4 Fast Flow beads and determined for SOD activity by modified pyrogallol autoxidation. Results Restriction analysis and sequencing proved that recombinant plasmid pGEX-4T-sod was constructed correctly. The expressed fusion protein SOD- GST, with a relative molecular mass of about 49 000, contained about 30. 2% of total somatic protein and existed in both superuatant and precipitate of lysate of recombinant E. coll. The purity and concentration of purified fusion protein were 82. 4% and 2. 8 mg/ml respectively. The specific activity of SOD purified from soluble expressed product was 1 440 U/nag. Conclusion The SOD of S. platensis, with bioactivity, was successfully expressed in E. coli and purified, which laid a foundation of large-scale production.