fibrinolytic activity of the fibrinolytic enzyme purified from sipunculus nudus

Purifying a Fibrinolytic Enzyme from &lt;em&gt;Sipunculus nudus&lt;/em&gt; via Affinity Chromatography

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&#39; plasminogen into active plasmin to degrade fibrin. Thus, their thrombolytic efficiency is heavily restricted by the patients&#39; 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.

Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus  

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

Our research is mainly about the purification of fibrinolytic enzyme. This work tries to establish a simple purification method for fibrinolytic enzyme. In this work, we report an arginine or lysine best-affinity purification system of fibrinolytic enzyme for the first time.Compared with the conventional purification methods, this is a simple, inexpensive, and efficient approach for the purification of fibrinolytic enzymes from Sipunculus nudus. The development of this protocol is of greater significance for the further utilization of fibrinolytic enzymes from Sipunculus nudus and other similar agents.

Watch this Scientific Journal Video about Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from  Sipunculus nudus at JoVE.com

Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from  Sipunculus nudus  

Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from Sipunculus nudus

Begin the fibrin plate preparation by mixing 25 milligrams of fibrinogen with 1.25 milliliters of physiological saline. Next, prepare the thrombin solution by thoroughly mixing 100 units of thrombin with 1.05 milliliters of physiological saline. Then, prepare the agarose solution by adding 0.5 grams of agarose to 22.5 milliliters of 0.02 molar Tris-hydrochloride acid buffer.Heat the agarose solution at 100 degrees Celsius until it dissolves completely. Cool the agarose solution to around 50 degrees Celsius before adding the fibrinogen solution to it. Immediately add the thrombin solution, mix rapidly, and pour the mixture into a 60-millimeter culture dish.To load the sample, punch three-millimeter wells into the fibrin plates using a sterile Boer, and fill them with 10 microliters of samples. Incubate the plates at 37 degrees Celsius for 18 hours before measuring the size of their degrading zones and calculating the fibrinolytic activity. Fibrinolytic activity evaluation showed a 1.3-centimeter lysing zone in the urokinase control, no lysing zone in the physiological saline buffer, 2.1 centimeters of the lysing zone in the crude protein well, and 1.8 centimeters diameter of the lysing zone in the purified fibrinolytic enzyme well.

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Research

JoVE Journal

Biochemistry

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&#160;19,200 U/mL,&#160;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.

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

Protein Isolation from Sipunculus nudus

Protein Isolation from Sipunculus nudus  

Begin the sample treatment by dissecting 100 grams of fresh Sipunculus nudus and collecting their intestine and inner fluid.Add 300 milliliters of Tris hydrochloride buffer and homogenize at 1000 RPM for 60 seconds.Freeze thaw the homogenate three times.After centrifuging the freeze thawed homogenate, collect the supernatant and store it at 4

Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts 

Filter the crude proteins extracted from the Sipunculus nudus intestine through a 0.22-micrometer filter membrane. Store this sample at four degrees Celsius for later use. Pack the arginine agarose matrix affinity chromatography column by loading the arginine agarose matrix medium into a five-milliliter empty chromatography column.Similarly, load the lysine agarose matrix affinity chromatography column by loading the lysine agarose matrix medium into an empty chromatography column. Equilibrate the affinity chromatography columns with double-distilled water followed by tris hydrochloride buffer. Load the filtered protein sample onto the pre-equilibrated column.Wash the column with five volumes of 0.02-molar tris hydrochloride buffer before eluting it with 0.15-0.25-0.35-0.45-0.55-and 0.65-molar sodium chloride solutions, successively. Once the elution peak appears in the 0.15-molar sodium chloride elution, collect the fractions in five-milliliter tubes. Concentrate the elution sample using a three-kilodalton ultracentrifugal filter and store this sample at minus 80 degrees Celsius for later use.When the protein solution was loaded onto the arginine agarose matrix affinity column, the flow-through peak appeared from approximately 40 to 66 minutes. In the gradient elution stage, elution with 0.15-molar sodium chloride peaked from approximately 94 to 110 minutes. When the protein solution was loaded to the lysine agarose matrix affinity column, the flow-through peak appeared from approximately 38 to 60 minutes.In the gradient elution stage, elution with 0.15-molar sodium chloride peaked from 80 to 86 minutes.