Published: June 2nd, 2023
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 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 19,200 U/mL, 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.
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' plasminogen into active plasmin to degrade fibrin. Thus, their thrombolytic efficiency is heavily restricted by the patients' plasminogen status3,4. Fibrinolytic agents, such as metalloproteinase plasmin and serine plasmin, are another type of clinical thromboly....
Following this protocol, crude tissue lysates were extracted, arginine-agarose matrix and lysine-agarose matrix affinity chromatography columns were built, purified sFE was obtained, and the purity and fibrinolytic activity of the purified sFE were measured by SDS-PAGE and fibrin plates, respectively.
After centrifugation, the collected supernatant was a transparent tan viscous liquid. Precipitation started when this supernatant was mixed with saturated ammonium sulfate solution (nine volumes).......
Due to the unavailability of the exact gene sequence of sFE, the currently used sFE was extracted from fresh S. nudus14. Moreover, the purification procedures of sFE reported in the literature were complicated and costly, as they were based on some general features of sFE, such as molecular weight, isoelectric point, ionic strength, and polarity15,16. No affinity purification protocol of sFE has been reported to date. In this stud.......
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