hHO—1结构预测及突变体的构建、表达、纯化和活性检测

hHO-1 STRUCTURE PREDICTION AND ITS MUTANT CONSTRUCT,EXPRESSION,PURIFICATION AND ACTIVITY ANALYSIS

人血红素加氧酶-1(human heme oxygenase-1,hHO-1)是血红素代谢的限速酶,直接调节体内胆红素水平。利用软件SPdbv程序对hHO-1进行结构模拟预测,以Ala取代第25位His残基,hHO-1活性部位的结构有较明显的改变,但据模拟推测突变后hHO-1与血红素仍然具有结合性。依据模拟结果,构建野生型和突变体表达载体pBHO-1和pBHO-1(M),并分别转化大肠杆菌DH5α,IPTG诱导表达目的蛋白。表达产物经30%-60%(NH_4)_2SO_4盐析后纯度提高3.6倍,再经两次Q-Sepharose Fast Flow阴离子交换树脂分离则表达产物的纯度提高30倍。酶活性测定显示,突变体hHO-1(△hHO-1)较野生型hHO-1(whHO-1)活性下降了91.21%。本研究显示hHO-1的第25位His在酶与底物血红素氧化反应中起着重要作用,为有效调控酶活性发挥其生物学作用提供依据。

Human Heme Oxygenase-l(hHO-1)is the rate-limiting enzyme in the catabolism re- action of heine,which directly regulates the concentration of bilirubin in human body. The mutant structure was simulated by Swiss-pdbviewer procedure,which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain,but the mutated enzyme still binded with heme.On the basis of the results, the expression vectors,pBHO-1 and pBHO-I(M),were constructed,induced by IPTG and expressed in E.coli DH5α strain.The ex- pression products were purified with 30%-60% saturation (NH_4)_2SO_4 and Q-Sepharose Fast Flow column chromatography.The concentration of hHO-1 in 30%-60% saturation (NH_4)_2SO_4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product,respectively.The activity of wild hHO-1(whHO-1) and mutant hHO-1(△hHO- 1)showed that the activity of △hHO-1 was reduced 91.21% compared with that of whHO-1.The study shows that His25 is of importance for the mechanism of hHO-1,and provides the possibility for effectively regulating the activity to exert biological function.