来源于豆豉的纳豆激酶基因在大肠杆菌中活性表达研究

Studies on active expression of nattokinase gene in E.coli

目的构建大肠杆菌表达质粒pTYB10 2 ,实现纳豆激酶基因 (nattokinasegene)在大肠杆菌中高活性表达。方法以纳豆芽孢杆菌基因组DNA为模板 ,采用PCR方法分别扩增编码前肽、成熟肽的序列 (pro NK) ,构建大肠杆菌表达质粒pTYB10 2 ,转化大肠杆菌ER2 5 6 6。在IPTG诱导下 ,分别在 37℃ (2h)、30℃ (3h)和 15℃ (14h)培养。结果pTYB10 2能表达有活性的纳豆激酶。SDS PAGE表明 ,15℃表达杂蛋白质更少。结论证明具有 77个氨基酸序列的前肽 (pro序列 )对纳豆激酶的活性表达是必不可少的。

PurposeThe expression vector pTYB102 was constructed and active expression of nattokinase gene in E. coli was realized.MethodsChromosome DNA was isolated from Bacillus natto. Pro-nk (encoding propeptide and mature peptide) was then amplified from chromosome by PCR. The expression vector pTYB102 was constructed and transformed into E.coli ER2566. The strain harboring pTYB102 can express active nattokinase under the induction of IPTG at 37℃(2 h), 30℃(3 h) or 15℃(14 h).ResultsSDS-PAGE shows that the proportion of expressed heteroprotein at 15℃ was lower than that at 30℃.ConclusionIt is confirmed that pro-sequence is indispensable for the active expression of nattokinase gene.