Name: Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts
Uploaded: 2023-06-02T12:10:00
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affinity chromatography mediated fibrinolytic enzymes purification from crude protein extracts 

Zuivering van een fibrinolytisch enzym uit Sipunculus nudus via affiniteitschromatografie

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.

Auteur in de schijnwerpers: Affiniteitszuivering van een fibrinolytisch enzym van Sipunculus nudus  

Affiniteitszuivering van een fibrinolytisch enzym van 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 Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts at JoVE.com

Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts   

Affiniteitschromatografie gemedieerde fibrinolytische enzymen Zuivering van ruwe eiwitextracten 

Filter de ruwe eiwitten geëxtraheerd uit de Sipunculus nudus darm door een 0,22-micrometer filtermembraan. Bewaar dit monster bij vier graden Celsius voor later gebruik. Verpak de arginine agarose matrix affiniteitschromatografiekolom door het arginine agarose matrixmedium in een lege chromatografiekolom van vijf milliliter te laden.Laad op dezelfde manier de kolom lysine-agarosematrixaffiniteitschromatografie door het lysine-agarosematrixmedium in een lege chromatografiekolom te laden. Breng de affiniteitschromatografiekolommen in evenwicht met dubbel gedestilleerd water gevolgd door trishydrochloridebuffer. Laad het gefilterde eiwitmonster op de vooraf gebalanceerde kolom.Was de kolom met vijf volumes 0,02-molaire trishydrochloridebuffer voordat u deze achtereenvolgens met 0,15-0,25-0,35-0,45-0,55- en 0,65-molaire natriumchlorideoplossingen elueert. Zodra de elutiepiek verschijnt in de 0,15-molaire natriumchloride-elutie, verzamelt u de fracties in buizen van vijf milliliter. Concentreer het elutiemonster met een ultracentrifugaalfilter van drie kilodalton en bewaar dit monster bij min 80 graden Celsius voor later gebruik.Toen de eiwitoplossing op de arginine agarose matrix affiniteitskolom werd geladen, verscheen de doorstroompiek van ongeveer 40 tot 66 minuten. In de gradiëntrelutiefase piekte de elutie met 0,15-molair natriumchloride van ongeveer 94 tot 110 minuten. Toen de eiwitoplossing in de affiniteitskolom van de lysine-agarosematrix werd geladen, verscheen de doorstroompiek van ongeveer 38 tot 60 minuten.In de gradiëngetalelutiefase piekte de elutie met 0,15-molair natriumchloride van 80 tot 86 minuten.

Research

JoVE Journal

Biochemistry

Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts 

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 ...