Nattokinase Gene Cloning And Expression

Natto is the Japanese traditional fermented food and has a very long history of at least 2, 000 years. Nattokinase is a kind of fibrinolytic enzymes discovered by Dr. Hiroyuki Sumi, in 1987. It is a serineproteinase, produced by Bacillus subtilis(natto). Attentions have been paid on it for its advantages of super ability of dissolving thrombus, safety, long half-time period and being effective by oral administration.Nattokinase is composed of 275 amino acids, its relative molecular weight is calculated to be 27728 according to the sequence of its' amino acids. The pre-pro-Nattokinase is composed of 381 amino acid residues, and the active form of 275 amino acid residues is formed by cutting 106 amino acid residues in the N side during the secretion process. The isoelectric point of Nattokinase is 8.6±0.3. It is comparatively stable in the normal pH and temperature.Recently, a series of researches to explore the gene sequence and the physiological and bio-chemical characteristics have been done, which showed that Nattokinase can be a new type of medicine to cure lysis thrombus. Nowadays, Nattokinase is by far the most potential fibrinolytic enzyme in more than 100 kinds of fibrinolytic substances discovered by human beings.At present, most of the studies have been done on strain improvement and fermentation condition optimization to increase Nattokinase productivity as well as on the purification of Nattokinase.In this research, genome DNA of Bacillus subtilis(natto) was extracted, used as the PCR template, two DNA segments of 1072 bp and 1194 bp, respectively, were cloned using PCR. Then the two gene segments were cloned into the vector of pMD18-T, transferred into the host strain, multiplied, purified, and analyzed using electrophoresis and sequencing, to confirm the two gene segments were the gene of Nattokinase. The gene expression vectors of pET22b-nk, pGEX-4T-1-nk for Nattokinase expression were constructed, transferred into E. coli BL21 (DE3), and expressed. The activities of the expressed Nattokinase were confirmed.