Abstract: The pro-nattokinase (pro-NK) gene, 1056bp, was amplified from genomic DNA of Bacillus subtilis by PCR.The amplified pro-NK gene was cloned into the expression vector pET-28a to construct the expression vector pET-28a-pro-NK.The recombinant vector pET-28a-pro-NK was transformed into E.coli BL21 (DE3) , and the protein of mature NK was efficiently expressed with IPTG induction.The SDS-PAGE analysis showed that the expressed NK was about 28ku, which was consistent with the theoretical calculation.The specific activity of mature NK purified by Ni2+ affinity chromatography was 34245.1IU/mg, however, declined to 16271.5IU/mg after the treatments of dialysis and freeze-drying without protectants.The protectant was optimized to keep the activity of mature NK in high level, indicating that the best ratios of the mass of the Mannitol to nattokinase were 1:2, increased by 30.9% compared with the treatments without protectants.The research provided a reference for producing pure nattokinase by genetic engineering, stabilizing the activity, and developing the clinical medicine.