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Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

Published: June 2nd, 2023



1Medicine School, Huaqiao University, 2Engineering Research Centre of Molecular Medicine of Ministry of Education

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....

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1. Preparation

  1. Sample treatment
    1. Carefully dissect fresh S. nudus (100 g) and collect the intestine and its inner fluid.
    2. Add 300 mL of Tris-HCl buffer (0.02 M, pH 7.4) for homogenization (1,000 rpm, 60 s).
    3. Freeze-thaw the homogenate 3x.
    4. Centrifuge the sample (10,956 × g, 0.5 h, 4 °C) and collect the supernatant. Store the sample at 4 °C until further use.
  2. Protein precipitation
    1. Mix .......

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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).......

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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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This research was funded by the Science and Technology Bureau of Xiamen City (3502Z20227197) and the Science and Technology Bureau of Fujian Province (No. 2019J01070, No.2021Y0027).


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NameCompanyCatalog NumberComments
30% Acrylamide-Bisacrylamide (29:1)Biosharp
Agarose G-10Biowest
Ammonium persulfateSINOPHARM
Ammonium sulfateSINOPHARM
Arginine-Sepharose 4BSolarbioArginine-agarose matrix
Bromoxylenol Blue (BPB)Solarbio
Fast Silver Stain KitBeyotime
Hydrochloric acidSINOPHARM
Lysine-Sepharose 4BSolarbioLysine-agarose matrix
N,N,N',N'-Tetramethylethylenediamine (TEMED)Sigma-Aldrich
Prestained Color Protein Marker (10-170 kD)Beyotime
Sodium chlorideSINOPHARM
Sodium Dodecyl Sulfonate (SDS)Sigma-Aldrich
Sodium hydroxideSINOPHARM
Tris(Hydroxymethyl) AminomethaneSolarbio
Tris(Hydroxymethyl) Aminomethane HydrochlorideSolarbio
AKT Aprotein Purification System pureGE
Automatic Vertical Pressure Steam Sterilizer MLS-3750SANYO
Chemiluminescence Imaging SystemGE
Constant Flow Pump BT-100QITE
Constant Temperature IncubatorJINGHONG
Desktop Refrigerated Centrifuge 3-30KSSIGMA
DHG Series Heating and Drying Oven DGG-9140ADSENXIN
Electric Glass Homogenizer DY89-IISCIENTZ
Electronic Analytical BalanceDENVER
Electro-Thermostatic Water Bath DK-S12SENXIN
Horizontal Decolorization ShakerKylin-Bell
Ice Machine AF 103Scotsman
KQ-500E Ultrasonic CleanerShuMei
Magnetic StirrerZhi wei
Micro Refrigerated Centrifuge H1650-WCence
Microwave OvenGalanz
Milli-Q ReferenceMillipore
PipettorThermo Fisher Scientific
Precision Desktop pH MeterSartorious
Small-sized Vortex OscillatorKylin-Bell
Vertical Electrophoresis SystemBio-Rad
Consumable Material 
200 µL PCR Tube (200 µL)Axygene
Centrifuge Tube (1.5 mL)Biosharp
Centrifuge Tube (5 mL)Biosharp
Centrifuge Tube (50 mL)NEST
Centrifuge Tube (7 mL)Biosharp
Culture Dish (60 mm)NEST
Filter Membrane (0.22 µm)Millex GP
Pipette Tip (1 mL )KIRGEN
Pipette Tip (10 µL)Axygene
Pipette Tip (200 µL)Axygene
Special Indicator PaperTZAKZY
Ultra Centrifugal Filter Unit (15 mL 3 KDa)Millipore
Ultra Centrifugal Filter Unit (4 mL 3 KDa)Millipore
Universal pH IndicatorSSS Reagent

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