Denne forskning blev finansieret af Science and Technology Bureau of Xiamen City (3502Z20227197) og Science and Technology Bureau of Fujian-provinsen (nr. 2019J01070, nr. 2021Y0027).

1. Forberedelse
<ol><li>Prøve behandling<ol><li>Dissekere forsigtigt frisk S. nudus (100 g) og saml tarmen og dens indre væske.</li>
		<li>Tilsæt 300 ml Tris-HCl-buffer (0,02 M, pH 7,4) til homogenisering (1.000 o / min, 60 s).</li>
		<li>Frys-optø homogenatet 3x.</li>
		<li>Prøven centrifugeres (10,956 × g, 0,5 timer, 4 °C), og supernatanten opsamles. Prøven opbevares ved 4 °C, indtil den anvendes yderligere.</li>
	</ol></li>
	<li>Protein udfældning<ol><li>Supernatanten bla...

Purifying a Fibrinolytic Enzyme from Sipunculus nudus via Affinity Chromatography

<ol>
	<li>Rosell, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patients+with+COVID-19+have+elevated+levels+of+circulating+extracellular+vesicle+tissue+factor+activity+that+is+associated+with+severity+and+mortality-brief+report.">Patients with COVID-19 have elevated levels of circulating extracellular vesicle tissue factor activity that is associated with severity and mortality-brief report.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (2), 878-882 (2021).</li><li>Schultz, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis+and+thrombocytopenia+after+ChAdOx1+nCoV-19+vaccination.+The+New+England.">Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination. The New England.</a> Journal of Medicine. 384 (22), 2124-2130 (2021).</li><li>von Kaulla, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urokinase-induced+fibrinolysis+of+human+standard+clots.">Urokinase-induced fibrinolysis of human standard clots.</a> Nature. 184 (4695), 1320-1321 (1959).</li><li>Van de Werf, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+thrombolysis+with+tissue-type+plasminogen+activator+in+patients+with+evolving+myocardial+infarction.">Coronary thrombolysis with tissue-type plasminogen activator in patients with evolving myocardial infarction.</a> The New England Journal of Medicine. 310 (10), 609-613 (1984).</li><li>Schaller, J., Gerber, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasmin-antiplasmin+system:+structural+and+functional+aspects.">The plasmin-antiplasmin system: structural and functional aspects.</a> Cellular and Molecular Life Sciences. 68 (5), 785-801 (2011).</li><li>Ge, Y. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+antithrombotic+protease+from+marine+worm+Sipunculus+nudus.">A novel antithrombotic protease from marine worm Sipunculus nudus.</a> International Journal of Molecular Sciences. 19 (10), 3023 (2018).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+novel+fibrinolytic+enzyme+from+culture+supernatant+of+Pleurotus+ostreatus.">Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus.</a> Journal of Microbiology and Biotechnology. 24 (2), 245-253 (2014).</li><li>Choi, J. -. H., Sapkota, K., Kim, S., Kim, S. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starase:+A+bi-functional+fibrinolytic+protease+from+hepatic+caeca+of+Asterina+pectinifera+displays+antithrombotic+potential.">Starase: A bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential.</a> Biochimie. 105, 45-57 (2014).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fibrinolytic+protein+From+Pheretima+vulgaris:+purification,+identification,+antithrombotic+evaluation,+and+mechanisms+investigation.">A novel fibrinolytic protein From Pheretima vulgaris: purification, identification, antithrombotic evaluation, and mechanisms investigation.</a> Frontiers in Molecular Biosciences. 8, 772419 (2022).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant,+hypolipidemic+and+hepatic+protective+activities+of+polysaccharides+from+Phascolosoma+esculenta.">Antioxidant, hypolipidemic and hepatic protective activities of polysaccharides from Phascolosoma esculenta.</a> Marine Drugs. 18 (3), 158 (2020).</li><li>. Preparation and application of natural fibrinolytic enzyme from peanut worm Available from: <a target="_blank" href="https://patents.google.com/patent/CN109295042A/en">https://patents.google.com/patent/CN109295042A/en</a> (2019)</li><li>Li, W., Yuan, M., Wu, Y., Xu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+expressed+differentially+in+female+and+male+gametes+of+Sipunculus+nudus.">Identification of genes expressed differentially in female and male gametes of Sipunculus nudus.</a> Aquaculture Research. 51 (9), 3780-3789 (2020).</li><li>Ossipow, V., Laemmlii, U. K., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+renature+DNA-binding+proteins+separated+by+SDS-polyacrylamide+gel+electrophoresis.">A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis.</a> Nucleic Acids Research. 21 (25), 6040-6041 (1993).</li><li>Hsu, T., Ning, Y., Gwo, J., Zeng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+barcoding+reveals+cryptic+diversity+in+the+peanut+worm+Sipunculus+nudus.">DNA barcoding reveals cryptic diversity in the peanut worm Sipunculus nudus.</a> Molecular Ecology Resources. 13 (4), 596-606 (2013).</li><li>Abiko, Y., Iwamoto, M., Shimizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen-plasmin+system.+I.+Purification+and+properties+of+human+plasminogen.">Plasminogen-plasmin system. I. Purification and properties of human plasminogen.</a> The Journal of Biochemistry. 64 (6), 743-750 (1968).</li><li>Abiko, Y., Iwamoto, M., Shimizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen-plasmin+system.+II.+Purification+and+properties+of+human+plasmin.">Plasminogen-plasmin system. II. Purification and properties of human plasmin.</a> The Journal of Biochemistry. 64 (6), 751-757 (1968).</li><li>Wiman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity-chromatographic+purification+of+human+&#945;2-antiplasmin.">Affinity-chromatographic purification of human &#945;2-antiplasmin.</a> The Biochemical Journal. 191 (1), 229-232 (1980).</li><li>Sandbjerg Hansen, M., Clemmensen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+purification+and+characterization+of+a+new+fast-acting+plasmin+inhibitor+from+human+platelets.+Evidence+for+non-identity+with+the+known+plasma+proteinase+inhibitors.">Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors.</a> The Biochemical Journal. 187 (1), 173-180 (1980).</li><li>Pietrocola, G., Rindi, S., Nobile, G., Speziale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+human+plasma/cellular+fibronectin+and+fibronectin+fragments.">Purification of human plasma/cellular fibronectin and fibronectin fragments.</a> Fibrosis. 1627, 309-324 (2017).</li><li>Nabiabad, H. S., Yaghoobi, M. M., Javaran, M. J., Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+analysis+and+purification+of+human+recombinant+tissue+type+plasminogen+activator+(rt-PA)+from+transgenic+tobacco+plants.">Expression analysis and purification of human recombinant tissue type plasminogen activator (rt-PA) from transgenic tobacco plants.</a> Preparative Biochemistry and Biotechnology. 41 (2), 175-186 (2011).</li><li>Shearin, T. V., Pizzo, S. V., Gonzalez-Gronow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+abnormalities+of+human+plasminogen+isolated+from+synovial+fluid+of+rheumatoid+arthritis+patients.">Molecular abnormalities of human plasminogen isolated from synovial fluid of rheumatoid arthritis patients.</a> Journal of Molecular Medicine. 75 (5), 378-385 (1997).</li></ol>

På grund af utilgængeligheden af den nøjagtige gensekvens af sFE blev den aktuelt anvendte sFE ekstraheret fra frisk S. nudus14. Desuden var rensningsprocedurerne for sFE rapporteret i litteraturen komplicerede og dyre, da de var baseret på nogle generelle træk ved sFE, såsom molekylvægt, isoelektrisk punkt, ionstyrke og polaritet15,16. Ingen affinitetsrensningsprotokol for sFE er blevet rapporteret til dato. I denne unders...

Trombose er en stor trussel mod folkesundheden, især efter den globale covid-19-pandemi 1,2. Klinisk er mange plasminogenaktivatorer (PA'er), såsom vævstype plasminogenaktivator (tPA) og urokinase (UK), blevet anvendt i vid udstrækning som trombolytiske lægemidler. PA'er kan aktivere patienternes plasminogen til aktivt plasmin for at nedbryde fibrin. Således er deres trombolytiske effektivitet stærkt begrænset af patienternes plasminogenstatus 3,4. Fibrinolytiske midler, såsom metalloproteinaseplasmin og serinplasmin, er en anden type klinisk trombolytisk lægemiddel, der også inkluderer fibrinolytiske enzymer (FE), såsom plasmin, som kan opløse blodpropper direkte, men hurtigt inaktiveres af forskellige plasminhæmmere5. Efterfølgende er der rapporteret om en ny type fibrinolytisk middel, der kan opløse tromben ved ikke kun at aktivere plasminogenet i plasmin, men også nedbryde fibrinet direkte 6-det fibrinolytiske enzym fra den gamle jordnøddeorm Sipunculus nudus (sFE)6. Denne bifunktion giver sFE andre fordele i forhold til traditionelle trombolytiske lægemidler, især med hensyn til unormal plasminogenstatus. Sammenlignet med andre bifunktionelle fibrinolytiske midler 7,8,9 viser sFE flere fordele, herunder sikkerhed, i forhold til ikke-fødevareafledte midler til lægemiddeludvikling, især for orale lægemidler. Dette skyldes, at biosikkerheden og biokompatibiliteten af Sipunculus nudus er veletableret10.
I lighed med de andre naturlige fibrinolytiske midler isoleret fra mikroorganismer, regnorme og svampe er oprensningen af sFE fra S. nudus meget kompliceret og inkluderer flere trin, såsom vævshomogenisering, ammoniumsulfatudfældning, afsaltning, anionbytterkromatografi, hydrofob interaktionskromatografi og molekylær sigtning10,11,12. Et sådant rensningssystem afhænger ikke kun af dygtige færdigheder og dyre materialer, men kræver også flere dage for at fuldføre hele proceduren. Derfor er et simpelt rensningsprogram af sFE af stor betydning for den videre udvikling af sFE. Heldigvis to krystaller af sFE (PDB: 8HZP; FBF: 8HZO) er opnået med succes (se supplerende fil 1 og supplerende fil 2). Gennem strukturel analyse og molekylære dockingeksperimenter fandt vi, at den katalytiske kerne i sFE specifikt kunne binde til mål, der indeholder arginin eller lysinrester.
Heri blev et affinitetsrensningssystem foreslået for første gang baseret på krystalstrukturen af sFE. Ved at følge denne protokol kunne meget ren og meget aktiv sFE renses fra de rå ekstrakter i et enkelt affinitetsrensningstrin. Den protokol, der udvikles her, er ikke kun vigtig for den store fremstilling af sFE, men kan også anvendes til oprensning af andre fibrinolytiske midler.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30% Acrylamide-Bisacrylamide (29:1)</td>
			<td>Biosharp</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Solarbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose G-10</td>
			<td>Biowest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>SINOPHARM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>SINOPHARM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arginine-Sepharose 4B</td>
			<td>Solarbio</td>
			<td>Arginine-agarose matrix</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromoxylenol Blue (BPB)</td>
			<td>Solarbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Silver Stain Kit</td>
			<td>Beyotime</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Solarbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SINOPHARM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kinase</td>
			<td>RHAWN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine-Sepharose 4B</td>
			<td>Solarbio</td>
			<td>Lysine-agarose matrix</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained Color Protein Marker (10-170 kD)</td>
			<td>Beyotime</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>SINOPHARM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfonate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SINOPHARM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Meilunbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(Hydroxymethyl) Aminomethane</td>
			<td>Solarbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(Hydroxymethyl) Aminomethane Hydrochloride</td>
			<td>Solarbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AKT Aprotein Purification System pure</td>
			<td>GE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Vertical Pressure Steam Sterilizer MLS-3750</td>
			<td>SANYO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescence Imaging System</td>
			<td>GE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Flow Pump BT-100</td>
			<td>QITE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Temperature Incubator</td>
			<td>JINGHONG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Refrigerated Centrifuge 3-30KS</td>
			<td>SIGMA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DHG Series Heating and Drying Oven DGG-9140AD</td>
			<td>SENXIN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electric Glass Homogenizer DY89-II</td>
			<td>SCIENTZ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Analytical Balance</td>
			<td>DENVER</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-Thermostatic Water Bath DK-S12</td>
			<td>SENXIN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal Decolorization Shaker</td>
			<td>Kylin-Bell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice Machine AF 103</td>
			<td>Scotsman</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KQ-500E Ultrasonic Cleaner</td>
			<td>ShuMei</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stirrer</td>
			<td>Zhi wei</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Refrigerated Centrifuge H1650-W</td>
			<td>Cence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave Oven</td>
			<td>Galanz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Reference</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Desktop pH Meter</td>
			<td>Sartorious</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small-sized Vortex Oscillator</td>
			<td>Kylin-Bell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Consumable Material&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L PCR Tube (200 &#181;L)</td>
			<td>Axygene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (5 mL)</td>
			<td>Biosharp</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (50 mL)</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (7 mL)</td>
			<td>Biosharp</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dish (60 mm)</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Membrane (0.22 &#181;m)</td>
			<td>Millex GP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (1 mL )</td>
			<td>KIRGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (10 &#181;L)</td>
			<td>Axygene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (200 &#181;L)</td>
			<td>Axygene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Special Indicator Paper</td>
			<td>TZAKZY</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Centrifugal Filter Unit (15 mL 3 KDa)</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Centrifugal Filter Unit (4 mL 3 KDa)</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Universal pH Indicator</td>
			<td>SSS Reagent</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

affinity purification of a fibrinolytic enzyme from sipunculus nudus

Det fibrinolytiske enzym fra Sipunculus nudus (sFE) er et nyt fibrinolytisk middel, der både kan aktivere plasminogen i plasmin og nedbryde fibrin direkte, hvilket viser store fordele i forhold til traditionelle trombolytiske midler. På grund af manglen på strukturel information er alle rensningsprogrammer for sFE imidlertid baseret på flertrinskromatografirensninger, som er for komplicerede og dyre. Her udvikles en affinitetsrensningsprotokol for sFE for første gang baseret på en krystalstruktur af sFE; det omfatter fremstilling af råprøven og lysin/arginin-agarosematrix-affinitetskromatografikolonnen, affinitetsoprensning og karakterisering af den rensede sFE. Efter denne protokol kan et parti sFE renses inden for 1 dag. Desuden øges renheden og aktiviteten af den rensede sFE til henholdsvis 92% og 19.200 E / ml. Dette er således en enkel, billig og effektiv tilgang til sFE-rensning. Udviklingen af denne protokol er af stor betydning for den videre anvendelse af sFE og andre lignende midler.

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.

Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus  

Affinitetsoprensning af et fibrinolytisk enzym fra Sipunculus nudus

Efter denne protokol blev råvævslysater ekstraheret, arginin-agarosematrix og lysin-agarosematrixaffinitetskromatografikolonner blev bygget, renset sFE blev opnået, og renheden og fibrinolytisk aktivitet af den rensede sFE blev målt med henholdsvis SDS-PAGE og fibrinplader.
Efter centrifugering var den opsamlede supernatant en gennemsigtig, solbrun tyktflydende væske. Udfældningen begyndte, da denne supernatant blev blandet med mættet ammoniumsulfatopløsning (ni rumfang). Efter at have...

Watch this Scientific Journal Video about Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus at JoVE.com

Her præsenterer vi en affinitetsrensningsmetode af et fibrinolytisk enzym fra Sipunculus nudus , der er enkelt, billigt og effektivt.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

Vores forskning handler primært om oprensning af fibrinolytisk enzym. Dette arbejde forsøger at etablere en simpel oprensningsmetode for fibrinolytisk enzym. I dette arbejde rapporterer vi for første gang et arginin- eller lysin-affinitetsrensningssystem af fibrinolytisk enzym.Sammenlignet med de konventionelle oprensningsmetoder er dette en enkel, billig og effektiv tilgang til oprensning af fibrinolytiske enzymer fra Sipunculus nudus. Udviklingen af denne protokol er af større betydning for den videre anvendelse af fibrinolytiske enzymer fra Sipunculus nudus og andre lignende midler.