重组蛹虫草超氧化物岐化酶脱辅基蛋白的表达、纯化及其活性重建

目的表达并纯化重组蛹虫草超氧化物歧化酶脱辅基蛋白,并考查各种金属离子对其活性重建的作用。方法用不含Cu2+和Zn2+的培养基培养重组菌株,并表达重组蛋白,采用DEAE-FF和CM-52进行纯化;在脱辅基蛋白溶液中加入Cu2+、Mn2+、Mg2+、Ni2+或Ag+,测定SOD酶活力的变化。结果蛹虫草脱辅基Cu,Zn-SOD在不含Cu2+和Zn2+的培养基中得到表达,纯化后的样品经SDS-PAGE分析,在相对分子质量17000处出现单一条带,经活性电泳分析无酶活力;脱辅基蛋白活性重建的最适宜Cu2+浓度为0.005mmol/L,但其紫外吸收图谱与天然SOD不同;0.1mmol/L的Mn2+、Mg2+、Ni2+、Ag+重建的酶活力远低于Cu2+重建的cm-SOD的酶活力。结论已成功表达并纯化了蛹虫草脱辅基Cu,Zn-SOD,各种金属离子只能有限地重建脱辅基Cu,Zn-SOD的活性。

Expression and Activity Reconstitution of Recombinant Cordyceps militaris Superoxide Dismutase Apoprotein

Objective To express and purify recombinant Cordyceps militaris superoxide dismutase (SOD) apoprotein and explore the reconstitution of activity of expressed product by metal ions. Methods Culture recombinant E. coli BL21(DE3) with plasmid pET-cm-SOD in copper ion- and zinc ion-free medium, and purify the expressed protein by DEAE-FF and CM-52 ion exchange chromatography. Add copper, manganese, magnesium, nickel or silver ion into the solution of expressed protein and determine the change of SOD activity. Results Apo-cm-SOD was expressed by using copper ion- and zinc ion-free medium. The expressed product showed a single band with relative molecular mass of 17 000 on SDS-PAGE profile, but no SOD activity by native PAGE. The optimal copper ion concentration for reconstitution of SOD activity was 0. 005 mmol/L. However, the ultraviolet absorption spectrum of expressed protein after SOD activity reconstitution was different from that of nature SOD. The SOD activities reconstituted by 0. 1 mmol/L manganese, magnesium, nickel and silver ions were significantly lower than that by copper ion. Conclusion Recombinant Cordyceps militaris SOD apoprotein was successfully expressed and purified. The SOD activity of expressed protein was only partially reconstituted by metal ions.