纳豆激酶酶原基因和纳豆激酶基因的克隆及在大肠杆菌中表达

Molecular clonging and expression of pro-nattokinase gene and nattokinase gene

目的:构建可高效生产有活性的纳豆激酶的大肠杆菌工程菌。方法:将纳豆激酶酶原(pro-nattokinase,pro-NK)基因和纳豆激酶(natokinase,NK)基因,并分别克隆到表达融合蛋白的高效表达载体pJN上,构建出表达质粒pJNK1和pJNK2,并转化大肠杆菌BL21(DE3)。结果:IPTG诱导下,两个融合蛋白的表达量均达到30%。活性检测显示表达纳豆激酶酶原融合蛋白的菌株pJNK-1(BL)诱导后菌体破碎上清的溶栓活性比表达纳豆激酶融合蛋白的菌株pJNK-2(BL)高2-3倍。结论:纳豆激酶酶原融合蛋白部分自减切产生纳豆激酶成熟肽。

Aim:To construct the engineered E.coli strain which expresses nattokinase with high expression level.Methods: Both pro-nattokinase gene and nattokinase gene were amplified and cloned into fusion expression vector pJN. Two expression plasmid pJNK1 and pJNK2 were constructed and transferred into BL21(DE3).Results: Both two fusion protein was expressed in a high expression level about 30% of the total cell protein. Preliminary biological assay result indicated the fibroinolytic effect of the induced product from pJNK1(BL) was 2-3 fold stronger than that from pJNK2(BL). Conclusion: Part of the pro-nattokinase fusion protein produced active natokinase through the intramolecular self-processing mechanism. 