金黄色葡萄球菌锰超氧化物歧化酶的表达纯化及活性分析

目的克隆表达、纯化金黄色葡萄球菌(Staphylococcus aureus)锰超氧化物歧化酶(Mn-SOD)并测定其活性。方法使用Clustal Omega比对不同来源Mn-SOD的序列,使用Swiss model网站预测金黄色葡萄球菌Mn-SOD的三级结构。提取细菌DNA,以此为模板PCR扩增获得Mn-SOD基因并将其与pET28a连接,构建pET28a-Mn-SOD重组质粒。将重组质粒转化大肠埃希菌BL21(DE3)获得重组菌BL/pET28a-Mn-SOD,使用IPTG诱导表达重组Mn-SOD,通过镍柱亲和层析纯化Mn-SOD蛋白并测定不同pH条件下的酶活性。结果金黄色葡萄球菌Mn-SOD具有Mn-SOD的特征序列和锰离子结合位点。成功得到重组质粒pET28a-Mn-SOD,转化大肠埃希菌BL21后表达相对分子质量为24.8×10^3的Mn-SOD,使用Ni^2+柱纯化后得到高纯度的Mn-SOD,且在pH值为7.5时活性较高,约为3 200 U/mL。结论成功构建了pET28a-Mn-SOD重组质粒能,表达的Mn-SOD重组蛋白纯化后仍具有SOD活性。

Expression,purification,and analysis of the activity of Staphylococcus aureus manganese superoxide dismutase

Objective To clone, express, and purify Staphylococcus aureus manganese superoxide dismutase(Mn-SOD) and determine its activity. Methods The amino acid sequences of Mn-SOD proteins from different sources were aligned using Clustal Omega, and the tertiary structure of S. aureus Mn-SOD was predicted using the Swiss model website. The Mn-SOD gene was obtained via amplification with PCR and ligated into pET28 a to construct a pET28 a-Mn-SOD recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 to obtain a recombinant BL/pET28a-Mn-SOD bacterium. Expression of the recombinant Mn-SOD protein was induced with IPTG. The protein was purified using nickel column affinity chromatography, and its enzyme activity under different pH conditions was determined. Results S. aureus Mn-SOD had a characteristic sequence of Mn-SOD protein and a manganese ion binding site. The recombinant plasmid pET28 a-Mn-SOD was successfully obtained and transformed into E. coli BL21(De3). It expressed an Mn-SOD protein with a molecular weight of 24.8×10^3. A highly pure Mn-SOD protein was obtained after purification with a Ni2^+ column. The Mn-SOD protein was highly active at a pH of 7.5, with activity of about 3,200 U/mL. Conclusion The recombinant plasmid pET28 a-Mn-SOD encodes a recombinant Mn-SOD protein, and the purified Mn-SOD protein had SOD activity.