Name: Protein Isolation from Sipunculus nudus
Uploaded: 2023-06-02T12:10:00
Description: Watch this Scientific Journal Video about Protein Isolation from Sipunculus nudus at JoVE.com

protein isolation from sipunculus nudus

Affinity Chromatography를 통한 Sipunculus nudus 의 Fibrinolytic Enzyme 정제

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.

저자 스포트라이트: Sipunculus nudus의 Fibrinolytic Enzyme의 친화성 정제  

Sipunculus nudus에서 섬유소 용해 효소의 친화성 정제

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 Protein Isolation from Sipunculus nudus at JoVE.com

Protein Isolation from Sipunculus nudus  

Sipunculus nudus에서 단백질 분리

신선한 Sipunculus nudus 100g을 해부하고 장과 내부 체액을 채취하여 샘플 처리를 시작합니다. 300 밀리리터의 트리스 하이드로 클로라이드 버퍼를 첨가하고 1000 RPM에서 60 초 동안 균질화합니다. 균질액을 세 번 동결 해동합니다. 동결 해동된 균질액을 원심분리한 후 상층액을 채취하여 4시에 보관한다

Research

JoVE Journal

Biochemistry

Protein Isolation 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 ...

연구

생화학

Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts 

친화성 크로마토그래피 매개 섬유소 용해 효소 조단백질 추출물 정제 

Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from Sipunculus nudus

Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from  Sipunculus nudus  

섬유소 용해 효소의 섬유소 용해 활성 Sipunculus nudus에서 정제