Name: Video: Protein Isolation from Sipunculus nudus
Uploaded: 2023-06-02T12:10:00
Description: 387 Views. Iniziare il trattamento del campione sezionando 100 grammi di Sipunculus nudus fresco e raccogliendo l'intestino e il liquido interno. Aggiungere 300 millilitri di tampone cloridrato Tris e omogeneizzare a 1000 giri/min per 60 secondi. Congelare, scongelare l'omogenato tre volte. Dopo aver centrifugato l'omogenato scongelato, raccogliere il surnatante e conservarlo a 4

protein isolation from sipunculus nudus

Purificazione di un enzima fibrinolitico da Sipunculus nudus mediante cromatografia di affinità

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.

Autore in primo piano: Purificazione per affinità di un enzima fibrinolitico da Sipunculus nudus  

Purificazione per affinità di un enzima fibrinolitico da Sipunculus nudus

Watch this Scientific Journal Video about Protein Isolation from Sipunculus nudus at JoVE.com

Protein Isolation from Sipunculus nudus  

Isolamento proteico da Sipunculus nudus

Iniziare il trattamento del campione sezionando 100 grammi di Sipunculus nudus fresco e raccogliendo l'intestino e il liquido interno. Aggiungere 300 millilitri di tampone cloridrato Tris e omogeneizzare a 1000 giri/min per 60 secondi. Congelare, scongelare l'omogenato tre volte. Dopo aver centrifugato l'omogenato scongelato, raccogliere il surnatante e conservarlo a 4

Research

JoVE Journal

Biochemistry

Protein Isolation from Sipunculus nudus

387 Views. Iniziare il trattamento del campione sezionando 100 grammi di Sipunculus nudus fresco e raccogliendo l'intestino e il liquido interno. Aggiungere 300 millilitri di tampone cloridrato Tris e omogeneizzare a 1000 giri/min per 60 secondi. Congelare, scongelare l'omogenato tre volte. Dopo aver centrifugato l'omogenato scongelato, raccogliere il surnatante e conservarlo a 4

Video: 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 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 ...