纳豆激酶原核表达载体的构建及其活性鉴定

利用引物搭桥的方法,经PCR扩增,获得了纳豆激酶成熟肽基因(NK),从而构建了大肠杆菌表达质粒NK/pTWIN1,NK/PET32a及NK/PML-c2x,经分别转化宿主菌ER2566,BL21和ER2566,获得了转纳豆激酶基因重组菌.结果表明,NK/pTWIN1-ER2566和NK/PET32a-BL21的纳豆激酶蛋白表达量较高.本研究选用NK/pTWIN1-ER2566作为表达菌株,对其进行发酵表达.SDS-PAGE显示,目的蛋白约占菌体总蛋白的36%,该蛋白是以包涵体形式存在的.包涵体经收集、蛋白变性及复性,并经过SP Sepharose分离纯化,得到了纯化的纳豆激酶蛋白,琼脂糖-纤维蛋白平板法测出1mg纳豆激酶干粉的溶栓活性相当于600u尿激酶.本文从基因工程角度研究纳豆激酶基因的克隆、表达及纯化,为利用基因工程菌生产纳豆激酶奠定了基础.图5表1参11

Expression and Purification of Nattokinase in E. Coli

Nattokinase gene was amplified using overlapping polymerase chain reaction (PCR), and cloned into pTWIN1, PET 32a and PML-c2x vectors. The expression plasmids of NK/pTWIN1, NK/PET32a and NK/PML-c2x were constructed and used to transform E. coli. The results showed that the level of the nattokinase protein was a little higher in E. coli transformed by NK/pTWIN1 and NK/PET32a. Nattokinase protein was purified by a method including protein denaturation, protein renaturation and SP sepharose from the E. coli ER2566 transformed by NK/pTWIN1. SDS-PAGE showed that the expression protein was about 36% of total cell protein. The experiment of aga-fibrous protein plate showed that the recombinant nattokinase had potent fibrinolytic activity equivalent to 600 urokinase units per milligram. The expression and purification of such a fibrinolytic enzyme would be helpful in producing nattokinase by gene engineering strain. Fig 5, Tab 1, Ref 11