Summary
Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.
Abstract
The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and 19,200 U/mL, respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.
Introduction
Thrombosis is a major threat to public health, especially following the Covid-19 global pandemic1,2. Clinically, many plasminogen activators (PAs), such as tissue-type plasminogen activator (tPA) and urokinase (UK), have been widely used as thrombolytic drugs. PAs can activate patients' plasminogen into active plasmin to degrade fibrin. Thus, their thrombolytic efficiency is heavily restricted by the patients' plasminogen status3,4. Fibrinolytic agents, such as metalloproteinase plasmin and serine plasmin, are another type of clinical thrombolytic drug that also include fibrinolytic enzymes (FE) such as plasmin, which can dissolve clots directly but are quickly inactivated by various plasmin inhibitors5. Subsequently, a novel type of fibrinolytic agent has been reported that can dissolve the thrombus by not only activating the plasminogen into plasmin but also degrading the fibrin directly6-the fibrinolytic enzyme from the ancient peanut worm Sipunculus nudus (sFE)6. This bifunction endows sFE other advantages over traditional thrombolytic drugs, especially in terms of abnormal plasminogen status. Compared with other bifunctional fibrinolytic agents7,8,9, sFE displays several advantages, including safety, over non-food derived agents for drug development, especially for oral drugs. This is because the biosafety and biocompatibility of Sipunculus nudus have been well-established10.
Similar to the other natural fibrinolytic agents isolated from microorganisms, earthworms, and mushrooms, the purification of sFE from S. nudus is very complicated and includes multiple stages, such as tissue homogenization, ammonium sulphate precipitation, desalination, anion-exchange chromatography, hydrophobic interaction chromatography, and molecular sieving10,11,12. Such a purification system not only depends on proficient skills and expensive materials, but also requires several days to complete the whole procedure. Therefore, a simple purification program of sFE is of great significance for the further development of sFE. Fortunately, two crystals of sFE (PDB: 8HZP; PDB: 8HZO) have been successfully obtained (see Supplemental File 1 and Supplemental File 2). Through structural analysis and molecular docking experiments, we found that the catalytic core of sFE could specifically bind to targets containing arginine or lysine residues.
Herein, an affinity purification system was proposed for the first time, based on the crystal structure of sFE. By following this protocol, highly pure and highly active sFE could be purified from the crude extracts in a single affinity purification stage. The protocol developed here is not only important for the large scale preparation of sFE, but also may be applied for the purification of other fibrinolytic agents.
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Protocol
1. Preparation
- Sample treatment
- Carefully dissect fresh S. nudus (100 g) and collect the intestine and its inner fluid.
- Add 300 mL of Tris-HCl buffer (0.02 M, pH 7.4) for homogenization (1,000 rpm, 60 s).
- Freeze-thaw the homogenate 3x.
- Centrifuge the sample (10,956 × g, 0.5 h, 4 °C) and collect the supernatant. Store the sample at 4 °C until further use.
- Protein precipitation
- Mix the supernatant with saturated ammonium sulfate solution (nine volumes) and let the mixture stand for 12 h at 4 °C.
NOTE: The protocol can be paused here and continued later. - Centrifuge (10,956 × g, 0.5 h, 4 °C) to obtain the protein precipitate; store it at 4 °C for later use.
- Mix the supernatant with saturated ammonium sulfate solution (nine volumes) and let the mixture stand for 12 h at 4 °C.
- Protein resuspension
- Resuspend the protein precipitate with 30 mL of Tris-HCl buffer (0.02 M, pH 7.4). Store this crude protein solution at 4 °C for later use.
2. Affinity chromatography
- Solution filtration
- Filter the crude protein solution through a 0.22 µm filter membrane. Store this sample at 4 °C for later use.
- Column packing
- Pack the arginine-agarose matrix affinity chromatography column and lysine-agarose matrix affinity chromatography column by loading an appropriate amount of lysine-agarose matrix medium and arginine-agarose matrix medium into 5 mL empty chromatography columns.
NOTE: The column can be stored at 2-8 °C, with the matrix immersed in store buffer (20% ethanol).
- Pack the arginine-agarose matrix affinity chromatography column and lysine-agarose matrix affinity chromatography column by loading an appropriate amount of lysine-agarose matrix medium and arginine-agarose matrix medium into 5 mL empty chromatography columns.
- Affinity purification
- Equilibrate the affinity chromatography column with ddH2O first (1 mL/min, 10 column volumes), followed by Tris-HCl buffer (0.02 M, pH 8.0, 1 mL/min, 5 column volumes).
- Load the protein solution sample onto the pre-equilibrated column (1 mL/min, 1/3 column volume).
NOTE: The volume of sample loading is dependent on the concentration of sFE. - Wash the column with Tris-HCl buffer (0.02 M, pH 8.0, five column volumes).
- Elute the column with 0.15 M, 0.25 M, 0.35 M, 0.45 M, 0.55 M, and 0.65 M NaCl (1 mL/min, five column volumes) successively.
- Look for the elution peak that should appear in the 0.15 M NaCl elution, and then collect the fractions in 5 mL tubes.
- Concentrate the elution sample using a 3 kD ultra centrifugal filter (3,944 × g, 1.5 h, 4 °C). Store this sample at -80 °C for later use.
3. Purity assessment
- Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation
- Prepare the SDS-PAGE gel according to Laemmli's method using 5% concentrated gel and 12% separation gel13.
- Electrophoresis
- Mix the sample (15 µL) with 5x SDS-PAGE protein loading buffer (1/4 volume), heat in boiling water for 5 min, load onto the SDS-PAGE gel, and run the gel at 80 V, 60 mA for 1.5 h.
- Analysis
- After electrophoresis, stain the gel with a Silver Stain Kit, according to the manufacturer's protocol, and observe the stained gel using a chemiluminescent imaging system.
- Analyze the purity of the target protein using the following formula: purity of target protein = intensity value of target protein/intensity value of total protein.
4. Fibrinolytic activity evaluation
- Fibrin plate preparation
- Prepare the fibrinogen solution by mixing 25 mg of fibrinogen with 1.25 mL of physiological saline.
- Prepare the thrombin solution by completely mixing 100 U of thrombin with 1.05 mL of physiological saline.
- Prepare the agarose solution by adding 0.5 g of agarose to 22.5 mL of Tris-HCl buffer (0.02 mol/L, pH 7.4). Mix and heat the agarose solution at 100 °C until dissolved completely.
- Cool the agarose solution down to around 50 °C and add fibrinogen solution. Then, immediately add the thrombin solution, mix them quickly, and pour them into a 60 mm culture dish.
- Loading
- Punch 3 mm wells on the prepared fibrin plates using a sterile borer and fill the wells with 10 µL samples.
- Analysis
- After 18 h of incubation at 37 °C, measure the fibrinolytic activity by calculating the size of their degrading zones. Fibrinolytic activity = (zone size of the sample/ zone size of urokinase) x 100 U. Zone size = diameter x diameter.
NOTE: Physiological saline buffer, crude protein (sample obtained in protocol step 1.3.1), and urokinase were used for the blank, negative control, and positive control, respectively. Compared to urokinase, we found that the fibrinolytic activity of the purified sFE was ~19,200 U/mL.
- After 18 h of incubation at 37 °C, measure the fibrinolytic activity by calculating the size of their degrading zones. Fibrinolytic activity = (zone size of the sample/ zone size of urokinase) x 100 U. Zone size = diameter x diameter.
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Representative Results
Following this protocol, crude tissue lysates were extracted, arginine-agarose matrix and lysine-agarose matrix affinity chromatography columns were built, purified sFE was obtained, and the purity and fibrinolytic activity of the purified sFE were measured by SDS-PAGE and fibrin plates, respectively.
After centrifugation, the collected supernatant was a transparent tan viscous liquid. Precipitation started when this supernatant was mixed with saturated ammonium sulfate solution (nine volumes). After standing for 12 h, a heavy precipitate was formed at the bottom of tube. When it was resuspended with Tris-HCl buffer, the protein precipitate disappeared quickly and dissolved in the buffer, appearing as a transparent pale-yellow liquid.
When the protein solution was loaded onto the arginine-agarose matrix affinity column, an obvious peak (peak 1, flowthrough peak) appeared at ~40 min and stopped eluting at ~66 min. In the gradient elution stage, when eluted with 0.15 M NaCl, an obvious peak (peak 2, elution peak) appeared at ~94 min and stopped eluting at ~110 min. No obvious elution peaks appeared in the remaining elution stages (0.25 M, 0.35 M, 0.45 M, 0.55 M, and 0.65 M NaCl) (Figure 1A). In this study, we reused the arginine-agarose matrix affinity column up to 10 times and found that the performance of the column was not significantly changed.
When the protein solution was loaded to the lysine-agarose matrix affinity column, an obvious peak (peak 1, flowthrough peak) appeared at ~38 min and stopped eluting at ~60 min. In the gradient elution stage, when eluted with 0.15 M NaCl, an obvious peak (peak 2, elution peak) appeared at ~80 min and stopped eluting at ~86 min. No obvious elution peaks appeared in the remaining elution stages (0.25 M, 0.35 M, 0.45 M, 0.55 M, and 0.65 M NaCl (Figure 1B). The elution profiles of the arginine-agarose matrix affinity column and lysine-agarose matrix affinity column were similar. As mentioned earlier, reusing the arginine-agarose matrix affinity column up to 10 times did not alter the performance of this column.
Purified sFE (10 µL, elution peak sample, peak 2) was added to the prepared fibrin plate. In addition, 10 µL of urokinase (100 U), 10 µL of physiological saline buffer, and 10 µL of crude protein (sample obtained in protocol step 1.3.1) were used for the positive control, blank, and negative control, respectively. After 18 h of incubation, two fibrin plates presented similar results: a lysing zone (1.3 cm diameter) appeared in the urokinase control; no lysing zone was observed in the physiological saline buffer; a lysing zone (2.1 cm diameter) appeared in the crude protein well; and a lysing zone (1.8 cm diameter) appeared in the purified sFE well (Figure 2). Compared to urokinase, we found that the fibrinolytic activity of the purified sFE was ~19,200 U/mL. As the elution peak sample was adjusted to the same volume as that of the sample loaded onto the affinity column, the recovery rate of the affinity column is indicated by the size (diameter x diameter) of the lysing zone on the fibrin plate. The recovery rate of the affinity column was ~73.5%.
The crude protein (sample obtained in protocol step 1.3.1) and purified sFE (elution peak sample, peak 2) were used for purity analysis. After SDS-PAGE and staining, three major protein bands (25, 26, and 27 kD) and six minor bands (20, 24, 28, 35, 40, and 45 kD) were presented in the crude protein. One major band (27 kD) and two minor bands (26 and 28 kD) were presented in the purified sFE (Figure 3). Through the gray value analysis, we found the purity of purified sFE was as high as ~92%.
Figure 1: The profile of affinity purification. (A) The crude sFE sample was purified by arginine-agarose matrix affinity purification. An obvious flowthrough peak appeared at ~40 min and stopped eluting at ~66 min. An obvious elution peak appeared at ~94 min and stopped eluting at ~110 min. (B) The crude sFE sample was purified by lysine-agarose matrix affinity purification. An obvious flowthrough peak appeared at ~38 min and stopped eluting at ~60 min. An obvious elution peak appeared at ~80 min and stopped eluting at ~86 min. Please click here to view a larger version of this figure.
Figure 2: Fibrinolytic activity evaluation. Samples were added to the fibrin plate and their fibrinolytic activity was measured and evaluated by their degrading zones on the plate after 18 h. Differently treated samples are indicated by red Arabic numerals. (A) The fibrinolytic activity of sFE purified by arginine-agarose matrix affinity column. #1, 100 U of urokinase; #2, physiological saline buffer; #3, crude protein (sample obtained in protocol step 1.3.1); #4, purified sFE (elution peak sample, peak 2). (B) The fibrinolytic activity of sFE purified by the lysine-agarose matrix affinity column. #1, 100 U of urokinase; #2, physiological saline buffer; #3, crude protein (sample obtained in protocol step 1.3.1); #4, purified sFE (elution peak sample, peak 2). Please click here to view a larger version of this figure.
Figure 3: Purity analysis. The purity of purified sFE was analyzed by SDS-PAGE. (A) The purity of sFE purified by the arginine-agarose matrix affinity column. M: protein marker; lane 1: crude protein (sample obtained in protocol step 1.3.1); lane 2: purified sFE (elution peak sample, peak 2). (B) The purity of sFE purified by the lysine-agarose matrix affinity column. M: protein marker; lane 1: crude protein (sample obtained in protocol step 1.3.1); lane 2: purified sFE (elution peak sample, peak 2). Please click here to view a larger version of this figure.
Supplemental File 1: 8ZHP: Crystal structure of snFPITE-n1. A PDB X-ray structural validation report. Please click here to download this File.
Supplemental File 2: 8ZHO: Crystal structure of snFPITE-n2. A PDB X-ray structural validation report. Please click here to download this File.
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Discussion
Due to the unavailability of the exact gene sequence of sFE, the currently used sFE was extracted from fresh S. nudus14. Moreover, the purification procedures of sFE reported in the literature were complicated and costly, as they were based on some general features of sFE, such as molecular weight, isoelectric point, ionic strength, and polarity15,16. No affinity purification protocol of sFE has been reported to date. In this study, an affinity purification protocol of sFE was successfully developed based on the knowledge of its crystal structure. Compared with the reported purification methods, this affinity purification method was easy to operate, inexpensive, and user-friendly. Moreover, a considerably higher recovery rate of the active sFE was observed by using this method.
Although many affinity purification methods of fibrinolytic enzymes have been reported, they were based on antibodies17, inhibitors18, ligands, and receptors16. The preparation of these antibodies, inhibitors, ligands, and receptors is technically challenging and laborious. Moreover, extra efforts are needed for conjugating these molecules to the matrix. In contrast, arginine-agarose matrix and lysine-agarose matrix are commercial materials, ready-to-use, highly stable, and easy to scale up19. Recently, lysine-agarose matrix has been used for the affinity purification of plasminogen20,21; however, plasminogen is the pro-enzyme of the plasmin, not an active form15. It remains uncertain whether lysine-agarose matrix is suitable for affinity purification of plasmin. Theoretically, the sFE purified by using arginine-agarose matrix and lysine-agarose matrix in this study is only the active form, because the crystal model has indicated that only the catalytic triads of sFE can interact with arginine and lysine residues. Therefore, this protocol could be applied in the purification of other serine plasmin, accelerating the development of other serine plasmin drugs.
Because the binding between sFE and the affinity column is not very strong, maintaining an exact NaCl concentration and proper elution speed are very critical for successful purification. Another key factor is the pH of the system, which severely affects the purification. These three parameters have been optimized by us. Admittedly, the recovery rate of active sFE in this study was relatively low, and may have been restricted by the low number of available arginine residues or lysine residues on the surface of the agarose matrix. Therefore, the linker between lysine/arginine and agarose matrix needs to be lengthened to enhance its recovery rate. Meanwhile, we could not exclude the possibility that other serine proteins with a similar catalytic triad as sFE will be eluted under this system. So, it is better to add a molecular weight-based purification technology, such as ultrafiltration, to separate the other serine proteins from sFE. Similarly, many other problems such as enhancing the binding ability, prolonging the lifetime, and further simplifying the procedure should be addressed in future work by according strategies (e.g., substituting the arginine/lysine with poly arginine/lysine and modification of the linker).
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Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgments
This research was funded by the Science and Technology Bureau of Xiamen City (3502Z20227197) and the Science and Technology Bureau of Fujian Province (No. 2019J01070, No.2021Y0027).
Materials
| Name | Company | Catalog Number | Comments |
| 30% Acrylamide-Bisacrylamide (29:1) | Biosharp | ||
| 2-Mercaptoethanol | Solarbio | ||
| Agarose G-10 | Biowest | ||
| Ammonium persulfate | SINOPHARM | ||
| Ammonium sulfate | SINOPHARM | ||
| Arginine-Sepharose 4B | Solarbio | Arginine-agarose matrix | |
| Bromoxylenol Blue (BPB) | Solarbio | ||
| Fast Silver Stain Kit | Beyotime | ||
| Fibrinogen | Merck | ||
| Glycine | Solarbio | ||
| Hydrochloric acid | SINOPHARM | ||
| Kinase | RHAWN | ||
| Lysine-Sepharose 4B | Solarbio | Lysine-agarose matrix | |
| N,N,N',N'-Tetramethylethylenediamine (TEMED) | Sigma-Aldrich | ||
| Prestained Color Protein Marker (10-170 kD) | Beyotime | ||
| Sodium chloride | SINOPHARM | ||
| Sodium Dodecyl Sulfonate (SDS) | Sigma-Aldrich | ||
| Sodium hydroxide | SINOPHARM | ||
| Thrombin | Meilunbio | ||
| Tris(Hydroxymethyl) Aminomethane | Solarbio | ||
| Tris(Hydroxymethyl) Aminomethane Hydrochloride | Solarbio | ||
| Equipment | |||
| AKT Aprotein Purification System pure | GE | ||
| Automatic Vertical Pressure Steam Sterilizer MLS-3750 | SANYO | ||
| Chemiluminescence Imaging System | GE | ||
| Constant Flow Pump BT-100 | QITE | ||
| Constant Temperature Incubator | JINGHONG | ||
| Desktop Refrigerated Centrifuge 3-30KS | SIGMA | ||
| DHG Series Heating and Drying Oven DGG-9140AD | SENXIN | ||
| Electric Glass Homogenizer DY89-II | SCIENTZ | ||
| Electronic Analytical Balance | DENVER | ||
| Electro-Thermostatic Water Bath DK-S12 | SENXIN | ||
| Horizontal Decolorization Shaker | Kylin-Bell | ||
| Ice Machine AF 103 | Scotsman | ||
| KQ-500E Ultrasonic Cleaner | ShuMei | ||
| Magnetic Stirrer | Zhi wei | ||
| Micro Refrigerated Centrifuge H1650-W | Cence | ||
| Microwave Oven | Galanz | ||
| Milli-Q Reference | Millipore | ||
| Pipettor | Thermo Fisher Scientific | ||
| Precision Desktop pH Meter | Sartorious | ||
| Small-sized Vortex Oscillator | Kylin-Bell | ||
| Vertical Electrophoresis System | Bio-Rad | ||
| Consumable Material | |||
| 200 µL PCR Tube (200 µL) | Axygene | ||
| Centrifuge Tube (1.5 mL) | Biosharp | ||
| Centrifuge Tube (5 mL) | Biosharp | ||
| Centrifuge Tube (50 mL) | NEST | ||
| Centrifuge Tube (7 mL) | Biosharp | ||
| Culture Dish (60 mm) | NEST | ||
| Filter Membrane (0.22 µm) | Millex GP | ||
| Parafilm | Bemis | ||
| Pipette Tip (1 mL ) | KIRGEN | ||
| Pipette Tip (10 µL) | Axygene | ||
| Pipette Tip (200 µL) | Axygene | ||
| Special Indicator Paper | TZAKZY | ||
| Ultra Centrifugal Filter Unit (15 mL 3 KDa) | Millipore | ||
| Ultra Centrifugal Filter Unit (4 mL 3 KDa) | Millipore | ||
| Universal pH Indicator | SSS Reagent |
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