Cloning Of Nattokinase Gene And Expression In Escherichia Coli And Schizosaccharomyces Pombe

Nattokinase(NK) is one kind of Bacillus subtilis protein kinase of the traditional fermented product, Natto, in Japan. Capable of being absorbed via oral, nattokinase has high efficiency in thrombi dissolving, and dissolves blood clots faster than traditional urokinase (UK) and streptokinase (SK), yet with longer half-life and less side effects. So nattokinase is a food-borne thrombolytic drug with great potential to be tapped, and it has aroused extensive concern in medical and food development.With an overall length of1146bp, the nattokinase gene(aprN) includes a complete open reading frame and coded381amino acids. The first29amino acid residues constitute a signal peptide, the following77ones constitute a propeptide, and the last275ones is a mature peptide. A pair of primers was designed based on the features of both aprN gene sequence and plasmid pET28a. On optimizing PCR conditions, the most favorable reaction condition was realized:pre-naturation at94℃for2min, denaturation at94℃for30s, annealing at55℃for30s, extension at72℃for1min. After repeating such operations for30times, the process was extended at72℃for10min. After the treatment, aprN gene with high specificity was obtained.The PCR product, aprN gene was cloned into the expressive vector pET28a. After being analyzed by restriction enzyme BamH I and Xho I, it was confirmed that exogenous segment subcloned into vector was aprN gene. The recombinant vector pET28a-aprN was transformed into strain E. coli BL21(DE3). Research on the growth rhythm of the recombinant strain showed that the introduction of aprN gene has little influence on the host cell. Research on induced expression of the recombinant strain suggested that the expression product’s fibrinolytic activity achieved its peak when the recombinant strain was cultured at20℃for20h with an IPTG addition of0.4mmol/L. The peak fibrinolytic activity can be up to81.73U/mL. A38KD protein band was detected through SDS-PAGE and this result agreed with what was expected.To further improve the stability and expression level of exogenous gene, the aprN gene was cloned into strain Schizosaccharomyces pombe. At first, a primer re-designed according to the features of the aprN gene and the plasmid, and PCR reaction conditions was explored, and probe for the nattokinase’s genetic fragment with better specificity was successfully constructed as recombinant plasmid pESP-2-aprN which was subsequently transformed into strain E. coli DH10B. Double digestion verification proves that the foreign gene cloned to the pESP-2plasmid was exactly the aprN gene. Use lithium acetate-based transformation was applied to transfer the recombinant plasmid into strain S. pombe yAS56, and the recombinant yeast strain was cultivated in selective medium. Colony PCR on the transformant was performed, and it is proved that the foreign gene has been transplanted into the yeast cells.Tests based on fibrin plate method manifests that the supernatant of cell lysate has little fibrinolytic activity. No fibrinolytic activity was found in the supernatant of cultivated cells. SDS-PAGE test indicated that the target protein was expressed only inside of cells and was not secreted outside of the cells. A protein band obviously tallying with expected protein was found in the cell lysate, while such a strip is never found in the supernatant.