Subtilisin QK (QK), which is highly homologous to nattokinase (NK), has comparatively high thrombolytic activity on both heated plasma and fibrin plate. The aim of this study was to produce QK in Pichia pastoris (P. pastoris) and establish improved techniques for its fermentation, purification and lyophilisation. QK was expressed as a secretory protein by P. pastoris; the thrombolytic activity of QK reached 112,000 IU (urokinase unit) per mL and the specific activity was 14,679 IU/mg under optimal culture and fermentation conditions. After purification by hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC), 91.73% of the thrombolytic activity of recombinant QK was recovered, the specific activity reached 16,272 IU/mg, and the purity of QK was 95%. Various agents were evaldated for their protective effects during freeze-drying at -50 degrees C. Trehalose and skim milk powder showed the highest lyoprotective effects. This study provides useful data regarding the potential use of QK in the treatment of cardiovascular diseases. (C) 2017 Elsevier Ltd. All rights reserved.