Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

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 pandemic^1,2. 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 status^3,4. 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 agents^7,8,9, 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 sieving^10,11,12. 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.

Protocol

1. Preparation

1. Sample treatment
   1. Carefully dissect fresh S. nudus (100 g) and collect the intestine and its inner fluid.
   2. Add 300 mL of Tris-HCl buffer (0.02 M, pH 7.4) for homogenization (1,000 rpm, 60 s).
   3. Freeze-thaw the homogenate 3x.
   4. Centrifuge the sample (10,956 × g, 0.5 h, 4 °C) and collect the supernatant. Store the sample at 4 °C until further use.
2. Protein precipitation
   1. 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.
   2. Centrifuge (10,956 × g, 0.5 h, 4 °C) to obtain the protein precipitate; store it at 4 °C for later use.
3. Protein resuspension
   1. 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

1. Solution filtration
   1. Filter the crude protein solution through a 0.22 µm filter membrane. Store this sample at 4 °C for later use.
2. Column packing
   1. 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).
3. Affinity purification
   1. 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).
   2. 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.
   3. Wash the column with Tris-HCl buffer (0.02 M, pH 8.0, five column volumes).
   4. 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.
   5. Look for the elution peak that should appear in the 0.15 M NaCl elution, and then collect the fractions in 5 mL tubes.
   6. 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

1. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation
   1. Prepare the SDS-PAGE gel according to Laemmli's method using 5% concentrated gel and 12% separation gel¹³.
2. Electrophoresis
   1. 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.
3. Analysis
   1. 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.
   2. 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

1. Fibrin plate preparation
   1. Prepare the fibrinogen solution by mixing 25 mg of fibrinogen with 1.25 mL of physiological saline.
   2. Prepare the thrombin solution by completely mixing 100 U of thrombin with 1.05 mL of physiological saline.
   3. 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.
   4. 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.
2. Loading
   1. Punch 3 mm wells on the prepared fibrin plates using a sterile borer and fill the wells with 10 µL samples.
3. Analysis
   1. 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.

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)...

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 polarity^15,16. No affinity purification protocol of sFE has been reported to date. In this stud...

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