JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

Published: June 2nd, 2023

DOI:

10.3791/65631

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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).......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
30% Acrylamide-Bisacrylamide (29:1)Biosharp
2-MercaptoethanolSolarbio
Agarose G-10Biowest
Ammonium persulfateSINOPHARM
Ammonium sulfateSINOPHARM
Arginine-Sepharose 4BSolarbioArginine-agarose matrix
Bromoxylenol Blue (BPB)Solarbio
Fast Silver Stain KitBeyotime
FibrinogenMerck
GlycineSolarbio
Hydrochloric acidSINOPHARM
KinaseRHAWN
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
ThrombinMeilunbio
Tris(Hydroxymethyl) AminomethaneSolarbio
Tris(Hydroxymethyl) Aminomethane HydrochlorideSolarbio
Equipment
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
ParafilmBemis
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

  1. Rosell, A., et al. Patients with COVID-19 have elevated levels of circulating extracellular vesicle tissue factor activity that is associated with severity and mortality-brief report. Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (2), 878-882 (2021).
  2. Schultz, N. H., et al. Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination. The New England. Journal of Medicine. 384 (22), 2124-2130 (2021).
  3. von Kaulla, K. N. Urokinase-induced fibrinolysis of human standard clots. Nature. 184 (4695), 1320-1321 (1959).
  4. Van de Werf, F., et al. Coronary thrombolysis with tissue-type plasminogen activator in patients with evolving myocardial infarction. The New England Journal of Medicine. 310 (10), 609-613 (1984).
  5. Schaller, J., Gerber, S. S. The plasmin-antiplasmin system: structural and functional aspects. Cellular and Molecular Life Sciences. 68 (5), 785-801 (2011).
  6. Ge, Y. -. H., et al. A novel antithrombotic protease from marine worm Sipunculus nudus. International Journal of Molecular Sciences. 19 (10), 3023 (2018).
  7. Liu, X., et al. Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus. Journal of Microbiology and Biotechnology. 24 (2), 245-253 (2014).
  8. Choi, J. -. H., Sapkota, K., Kim, S., Kim, S. -. J. Starase: A bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential. Biochimie. 105, 45-57 (2014).
  9. Liu, H., et al. A novel fibrinolytic protein From Pheretima vulgaris: purification, identification, antithrombotic evaluation, and mechanisms investigation. Frontiers in Molecular Biosciences. 8, 772419 (2022).
  10. Wu, Y., et al. Antioxidant, hypolipidemic and hepatic protective activities of polysaccharides from Phascolosoma esculenta. Marine Drugs. 18 (3), 158 (2020).
  11. . Preparation and application of natural fibrinolytic enzyme from peanut worm Available from: https://patents.google.com/patent/CN109295042A/en (2019)
  12. Li, W., Yuan, M., Wu, Y., Xu, R. Identification of genes expressed differentially in female and male gametes of Sipunculus nudus. Aquaculture Research. 51 (9), 3780-3789 (2020).
  13. Ossipow, V., Laemmlii, U. K., Schibler, U. A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis. Nucleic Acids Research. 21 (25), 6040-6041 (1993).
  14. Hsu, T., Ning, Y., Gwo, J., Zeng, Z. DNA barcoding reveals cryptic diversity in the peanut worm Sipunculus nudus. Molecular Ecology Resources. 13 (4), 596-606 (2013).
  15. Abiko, Y., Iwamoto, M., Shimizu, M. Plasminogen-plasmin system. I. Purification and properties of human plasminogen. The Journal of Biochemistry. 64 (6), 743-750 (1968).
  16. Abiko, Y., Iwamoto, M., Shimizu, M. Plasminogen-plasmin system. II. Purification and properties of human plasmin. The Journal of Biochemistry. 64 (6), 751-757 (1968).
  17. Wiman, B. Affinity-chromatographic purification of human α2-antiplasmin. The Biochemical Journal. 191 (1), 229-232 (1980).
  18. Sandbjerg Hansen, M., Clemmensen, I. Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors. The Biochemical Journal. 187 (1), 173-180 (1980).
  19. Pietrocola, G., Rindi, S., Nobile, G., Speziale, P. Purification of human plasma/cellular fibronectin and fibronectin fragments. Fibrosis. 1627, 309-324 (2017).
  20. Nabiabad, H. S., Yaghoobi, M. M., Javaran, M. J., Hosseinkhani, S. Expression analysis and purification of human recombinant tissue type plasminogen activator (rt-PA) from transgenic tobacco plants. Preparative Biochemistry and Biotechnology. 41 (2), 175-186 (2011).
  21. Shearin, T. V., Pizzo, S. V., Gonzalez-Gronow, M. Molecular abnormalities of human plasminogen isolated from synovial fluid of rheumatoid arthritis patients. Journal of Molecular Medicine. 75 (5), 378-385 (1997).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved