All methods were carried out in accordance with relevant guidelines and regulations.
All experimental protocols were approved by Zhongnan Hospital of Wuhan University.
Strains, plasmids, and media
E. coli XL10-Gold and P. pastoris GS115 were purchased from Invitrogen (USA). The vector pUC57 was purchased from GenScript (USA), and pHMB905BDM (d1 + 2 × 201 AOX1 promoter , MF4I signal sequence, Ampr) was constructed and stored in our lab. Luria-Bertani (LB) medium was prepared as described in the Manual of Molecular Cloning. Buffered glycerol-complex (BMGY) medium, buffered methanol-complex (BMMY) medium, and minimal dextrose (MD) medium were prepared as described in the instruction manual of the Invitrogen Pichia expression kit (USA) [29, 30]. BMMY medium supplemented with casein was prepared by dissolving 1% casein in phosphate buffer (pH 6.0), followed by the addition of BMMY ingredients and sterilization at 121 °C for 20 min. Lumbrokinase and fibrinogen were purchased from Sigma (USA).
Design and synthesis of the truncated NK-Bs-encoding open reading frame
A truncated nattokinase-encoding gene from B. subtilis (NK-Bs) was used for heterologous expression. The DNA sequence of NK-Bs, excluding the first 52-bp signal sequence, was modified to match the codon usage bias of P. pastoris (S1). This edited open reading frame (ORF) was synthesized by Genecreate (China) and cloned into pUC57 for DNA sequencing.
Construction of recombinant vectors for the expression of NK-Bs
The aprN gene of B. subtilis natto (Genbank accession number AEV91244.1) without the signal peptide coding sequence was optimized based on the codon preference of P. pastoris and synthesized. The DNA fragment was cloned into the expression vector pHBM905BDM to form an ORF encoding an NK-Bs protein fused with an N-terminal MF-4I leader sequence for secretory expression and a C-terminal 6× His-tag for affinity purification. It was amplified using the primers Natt-1 and Natt-2 (Table S1) and subsequently treated with T4 DNA polymerase in the presence of 1 mM deoxythymidine triphosphate (dTTP) for 20 min at 12 °C to form overhangs compatible with the sticky ends of pHBM905BDM digested with CpoI and NotI [29, 30]. These two fragments were ligated with T4 ligase (TaKaRa, China), followed by transformation into E. coli XL10-Gold and screening on LB plates supplemented with 100 μg/mL ampicillin. Biobrick assembly was used for the construction of multicopy nattokinase expression vectors.
Screening of recombinant yeast strains expressing NK-Bs
The recombinant plasmids were linearized using SalI and transformed into P. pastoris GS115 by electroporation (7000 V/cm, 25 μF, 400×; Life Technologies Cell-Porator, USA). Transformants were selected on MD plates without histidine, followed by identification on BMMY plates supplemented with 1% casein [29, 30].
Expression of recombinant NK-Bs using shake-flask fermentation
Recombinants bearing different copies of the target gene were inoculated into 50 mL BMGY medium and incubated at 28 °C for 2 days. The cells in each culture were collected by centrifugation at 4000×g for 5 min and individually inoculated into 30 mL BMMY medium. Then, 1% (v/v) methanol was added every 24 h to induce the expression of the foreign protein. Cell cultures were centrifuged at 10,000×g for 5 min at 4 °C after induction for 5 days, and the supernatants were collected.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein concentration measurements
Samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (w/v) polyacrylamide gel, followed by staining with Coomassie Brilliant Blue G-250. Protein concentrations were determined using the Bradford method with a BCA protein kit (Pierce, USA). A standard curve was created using bovine serum albumin with concentrations ranging from 0.1 to 0.6 mg/mL [29, 30].
Purification of the target protein
After induction with methanol for 120 h, the supernatant of the cell culture was collected and purified using Ni-NTA affinity chromatography. Approximately 20 mL of the supernatant was applied to 1 mL Ni-NTA beads. The column was washed twice with two column volumes of wash buffer (50 mM Tris-HCl; 200 mM NaCl; 50 mM NaH2PO4; 40 mM imidazole, pH 8.0). One column volume of elution buffer (50 mM Tris-HCl; 200 mM NaCl; 50 mM NaH2PO4; 200 mM imidazole, pH 8.0) was used to recover the target protein. The sample was then collected and dialyzed using a Millipore 10-kDa cut-off membrane at 4 °C to remove ions and salts, followed by resuspension in storage buffer (50 mM Tris-HCl, pH 7.5) [29, 30].
Analysis of the serine proteinase activity of NK-Bs
Enzyme activity was measured using casein as the substrate. Briefly, 1 mL of diluted enzyme sample was mixed with an equal volume of 2% (w/v) casein in 20 mM phosphate buffer (pH 9.0), followed by incubation at 65 °C for 10 min. The reaction was terminated using 2 mL of 10% (w/v) trichloroacetic acid. The mixture was centrifuged at 14,000×g for 10 min and the optical density at 280 nm (OD280) of the supernatant was measured to determine the amount of tyrosine released during the reaction. One unit of enzymatic activity was defined as the amount of enzyme needed to catalyze the release of 1 μg tyrosine per min at 65 °C and pH 9.0. All experiments were performed in triplicate .
Analysis of the characteristics of NK-Bs
To measure the optimal pH value for the recombinant enzyme, the pH of the enzyme solution was adjusted using the following buffers: sodium lactate (50 mM, pH 2.0–3.5); sodium acetate (50 mM, pH 3.5–6.0); phosphate buffer (50 mM, pH 6.0–7.0); Tris-HCl (50 mM, pH 7.0–9.0), or Na2B4O7/NaOH (50 mM, pH 9.0–12.0). The enzyme activity was then measured at 55 °C.
To determine the optimal temperature for the recombinant enzyme, the enzyme was diluted with Na2B4O7/NaOH buffer (50 mM, pH 10.5) to the appropriate concentration, and the reaction was carried out at a range of temperatures (50–80 °C, at 5 °C increments).
To investigate the thermostability of NK-Bs, the purified NK-Bs was divided into six samples and incubated at 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, or 75 °C. Exactly 1 mL of each sample was collected every 10 min, and the remaining enzyme activity of all samples was measured at pH 9.0 and 65 °C.
To investigate the pH stability of NK-Bs, purified NK-Bs was incubated at pH 2–13 for 60 min, and the activity of the remaining enzyme was measured at pH 9.0 and 65 °C. Each experiment was performed in triplicate.
Expression of recombinant NK-Bs using high-density fermentation
Fed-batch fermentation was performed according to the Invitrogen Pichia Fermentation Process Guidelines. The recombinant P. pastoris strain was inoculated into 200 mL YPD and cultivated at 28 °C for 24 h. The cell culture was then transferred to 2 L BSM medium in a 5-L fermenter (BaoXing, China). During the early stages of fermentation, the culture was maintained at 28 °C, pH 5.8, and 30% dissolved oxygen (DO). After approximately 18–24 h, glycerol was exhausted and DO was rapidly increased to 100%. To continue cell growth, 50% (v/v) glycerol supplemented with PTM trace salts (12 mL/L) was added at 12 mL/h/L, and DO was maintained above 20%. When the OD600 reached approximately 300, methanol containing PTM trace salts (12 mL/L) was fed at a speed of 3 mL/h/L to induce the expression of the target gene. The fermentation conditions were adjusted to 25 °C and pH 5.0, and DO was maintained at 20–30%. After 2 h, the feeding speed of methanol was increased by a ratio of 20% per hour until it reached 7 mL/h/L. These conditions were maintained until the end of fermentation [29, 30].
Analysis of the fibrinolytic activity of NK-Bs in vitro
Fibrinolytic activity was measured using the standard fibrin plate method with modifications . In a petri dish, 7.3 mL agarose (0.5% w/v) and 200 μL bovine thrombin (1 mg/mL) were gently mixed, followed by the addition of 2.5 mL bovine fibrinogen (1% w/v) to induce a solid fibrin formation. The fibrin plate was incubated at 37 °C for 18 h after 10 μL of the enzyme sample was injected into the bottom of the fibrin agar. The diameters of the clearing zones were used as indicators of the fibrinolytic activity of the enzyme.
Analysis of the fibrinolytic effect of NK-Bs in vivo
To test the thrombolytic activity of recombinant NK-Bs in vivo, a rat model of microvascular thrombosis was established . Female Sprague-Dawley rats weighing approximately 180–220 g were used. Thrombosis was induced by hypodermic injection of κ-carrageenan after the rats were fasted for 8 h. Thrombosis was evaluated by the length of tail infarction. The length of the infarcted region at the tip of the tail was measured 8, 12, and 24 h after injection. After the thrombosis model was established, the rats were split into six groups. Different dosages of the recombinant NK-Bs (50,000 IU/kg for high dose, 10,000 IU/kg for medium dose, and 2000 IU/kg for low dose) were fed to the rats every 12 h for 48 h. Vermis kinase, a known thrombolytic agent, was used as a positive control (10,000 IU/kg), and physiologic saline was used as the negative control. Each group included five duplicates.
Twelve hours after intragastric administration, the rats were anaesthetized by intraperitoneal injection with 50% ethyl carbamate at a dose of 5 mL/kg. Approximately 1.8 mL of blood from the postcaval vein was drawn into anticoagulant tubes containing 0.2 mL sodium citrate (0.109 mol/L). The blood in the tubes was centrifuged at 4000×g for 15 min, and 250 μL of the top layer of plasma was collected. Fibrin/fibrinogen degradation products (FDPs) and D-dimer levels were measured using the double antibody sandwich method (ABC-ELISA) according to the manufacturer’s protocol (Elabscience Biotechnology, China). Probabilities of less than 5% (P < 0.05) were considered statistically significant. All values are expressed as mean ± standard error of mean (S.E.M.) to show variation in groups.
Histological examination was carried out as previously described [8, 9, 33]. Twelve hours after the nattokinase treatment was completed, rats were euthanized by spinal dislocation, and ear tissue was soaked in a formaldehyde solution before preparation of pathological sections. The tissue samples were embedded with paraffin and stained with hematoxylin and eosin for observation under a Upright optical microscope (Nikon Eclipse E100, Nikon DS-U3, 400).