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. Begin the sample treatment by dissecting 100 grams of fresh Sipunculus nudus and collecting their intestine and inner fluid.

Add 300 milliliters of Tris-HCl buffer and homogenize at 1, 000 rpm for 60 seconds. Freeze-thaw the homogenate three times. After centrifuging the freeze-thawed homogenate, collect the supernatant and store it at four degrees Celsius until further use.

To begin the protein precipitation, remove the supernatant from four degrees Celsius and mix it with nine volumes of saturated ammonium sulfate solution. Incubate the mixture for 12 hours at four degrees Celsius. Then, precipitate the protein by centrifuging the mixture and store the precipitated protein at four degrees Celsius.

Resuspend the protein precipitate in 30 milliliters of Tris-HCl buffer and store this crude protein solution at four degrees Celsius for later use. 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-HCl buffer.

Load the filtered protein sample onto the pre-equilibrated column. Wash the column with five volumes of 0.02 molar Tris-HCl 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 3 kDa ultra centrifugal filter and store this sample at 80 degrees Celsius for later use. When the protein solution was loaded onto the arginine-agarose matrix affinity column, the flowthrough 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 flowthrough 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. 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-Hcl 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 borer 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.