fibrinolytic activity of the fibrinolytic enzyme purified from sipunculus nudus

Очищение фибринолитического фермента из Sipunculus nudus с помощью аффинной хроматографии

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.

В центре внимания автора: Аффинное очищение фибринолитического фермента из Sipunculus nudus  

Аффинная очистка фибринолитического фермента из 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  

Фибринолитическая активность фибринолитического фермента, очищенного от Sipunculus nudus

Начните приготовление фибриновой пластины с смешивания 25 миллиграммов фибриногена с 1,25 миллилитрами физиологического раствора. Далее приготовьте раствор тромбина, тщательно смешав 100 единиц тромбина с 1,05 миллилитрами физиологического раствора. Затем приготовьте раствор агарозы, добавив 0,5 грамма агарозы к 22,5 миллилитрам 0,02-молярного буфера трис-гидрохлоридной кислоты.Нагрейте раствор агарозы при 100 градусах Цельсия до полного растворения. Охладите раствор агарозы примерно до 50 градусов Цельсия, прежде чем добавлять в него раствор фибриногена. Сразу же добавьте раствор тромбина, быстро перемешайте и вылейте смесь в 60-миллиметровую культуральную чашку.Чтобы загрузить образец, пробивайте трехмиллиметровые лунки в фибриновые пластины с помощью стерильного бура и заполняйте их 10 микролитрами образцов. Инкубируйте пластины при температуре 37 градусов Цельсия в течение 18 часов, прежде чем измерить размер их зон разложения и рассчитать фибринолитическую активность. Оценка фибринолитической активности показала 1,3-сантиметровую зону лизинга в контроле урокиназы, отсутствие зоны лизинга в физиологическом солевом буфере, 2,1 сантиметра зоны лизинга в лунке сырого белка и диаметр зоны лизинга 1,8 сантиметра в лунке очищенного фибринолитического фермента.

Research

JoVE Journal

Biochemistry

Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from Sipunculus nudus

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

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  

Выделение белка из Sipunculus nudus

Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts 

Аффинная хроматография, опосредованная очисткой фибринолитических ферментов от экстрактов сырого белка 