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).

1. Preparation
<ol>
	<li>Sample treatment
	<ol>
		<li>Carefully dissect fresh S. nudus (100 g) and collect the intestine and its inner fluid.</li>
		<li>Add 300 mL of Tris-HCl buffer (0.02 M, pH 7.4) for homogenization (1,000 rpm, 60 s).</li>
		<li>Freeze-thaw the homogenate 3x.</li>
		<li>Centrifuge the sample (10,956 &#215; g, 0.5 h, 4 &#176;C) and collect the supernatant. Store the sample at 4 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Protein precipitation
	<ol>
		<li>Mix the supernatant with saturated ammonium sulfate solution (nine volumes) and let the mixture stand for 12 h at 4 &#176;C. 
		NOTE: The protocol can be paused here and continued later.</li>
		<li>Centrifuge (10,956 &#215; g, 0.5 h, 4 &#176;C) to obtain the protein precipitate; store it at 4 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Protein resuspension
	<ol>
		<li>Resuspend the protein precipitate with 30 mL of Tris-HCl buffer (0.02 M, pH 7.4). Store this crude protein solution at 4 &#176;C for later use.</li>
	</ol>
	</li>
</ol>
2. Affinity chromatography
<ol>
	<li>Solution filtration
	<ol>
		<li>Filter the crude protein solution through a 0.22 &#181;m filter membrane. Store this sample at 4 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Column packing
	<ol>
		<li>Pack the arginine-agarose matrix affinity chromatography column and lysine-agarose matrix affinity chromatography column by loading an appropriate amount of lysine-agarose matrix medium and arginine-agarose matrix medium into 5 mL empty chromatography columns. 
		NOTE: The column can be stored at 2-8 &#176;C, with the matrix immersed in store buffer (20% ethanol).</li>
	</ol>
	</li>
	<li>Affinity purification
	<ol>
		<li>Equilibrate the affinity chromatography column with ddH2O first (1 mL/min, 10 column volumes), followed by Tris-HCl buffer (0.02 M, pH 8.0, 1 mL/min, 5 column volumes).</li>
		<li>Load the protein solution sample onto the pre-equilibrated column (1 mL/min, 1/3 column volume). 
		NOTE: The volume of sample loading is dependent on the concentration of sFE.</li>
		<li>Wash the column with Tris-HCl buffer (0.02 M, pH 8.0, five column volumes).</li>
		<li>Elute the column with 0.15 M, 0.25 M, 0.35 M, 0.45 M, 0.55 M, and 0.65 M NaCl (1 mL/min, five column volumes) successively.</li>
		<li>Look for the elution peak that should appear in the 0.15 M NaCl elution, and then collect the fractions in 5 mL tubes.</li>
		<li>Concentrate the elution sample using a 3 kD ultra centrifugal filter (3,944 &#215; g, 1.5 h, 4 &#176;C). Store this sample at -80 &#176;C for later use.</li>
	</ol>
	</li>
</ol>
3. Purity assessment
<ol>
	<li>Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation
	<ol>
		<li>Prepare the SDS-PAGE gel according to Laemmli&#39;s method using 5% concentrated gel and 12% separation gel13.</li>
	</ol>
	</li>
	<li>Electrophoresis
	<ol>
		<li>Mix the sample (15 &#181;L) with 5x SDS-PAGE protein loading buffer (1/4 volume), heat in boiling water for 5 min, load onto the SDS-PAGE gel, and run the gel at 80 V, 60 mA for 1.5 h.</li>
	</ol>
	</li>
	<li>Analysis
	<ol>
		<li>After electrophoresis, stain the gel with a Silver Stain Kit, according to the manufacturer&#39;s protocol, and observe the stained gel using a chemiluminescent imaging system.</li>
		<li>Analyze the purity of the target protein using the following formula: purity of target protein = intensity value of target protein/intensity value of total protein.</li>
	</ol>
	</li>
</ol>
4. Fibrinolytic activity evaluation
<ol>
	<li>Fibrin plate preparation
	<ol>
		<li>Prepare the fibrinogen solution by mixing 25 mg of fibrinogen with 1.25 mL of physiological saline.</li>
		<li>Prepare the thrombin solution by completely mixing 100 U of thrombin with 1.05 mL of physiological saline.</li>
		<li>Prepare the agarose solution by adding 0.5 g of agarose to 22.5 mL of Tris-HCl buffer (0.02 mol/L, pH 7.4). Mix and heat the agarose solution at 100 &#176;C until dissolved completely.</li>
		<li>Cool the agarose solution down to around 50 &#176;C and add fibrinogen solution. Then, immediately add the thrombin solution, mix them quickly, and pour them into a 60 mm culture dish.</li>
	</ol>
	</li>
	<li>Loading
	<ol>
		<li>Punch 3 mm wells on the prepared fibrin plates using a sterile borer and fill the wells with 10 &#181;L samples.</li>
	</ol>
	</li>
	<li>Analysis
	<ol>
		<li>After 18 h of incubation at 37 &#176;C, measure the fibrinolytic activity by calculating the size of their degrading zones. Fibrinolytic activity = (zone size of the sample/ zone size of urokinase) x 100 U. Zone size = diameter x diameter. 
		NOTE: Physiological saline buffer, crude protein (sample obtained in protocol step 1.3.1), and urokinase were used for the blank, negative control, and positive control, respectively. Compared to urokinase, we found that the fibrinolytic activity of the purified sFE was ~19,200 U/mL.</li>
	</ol>
	</li>
</ol>

Purifying a Fibrinolytic Enzyme from Sipunculus nudus via Affinity Chromatography

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<li>Schaller, J., Gerber, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+plasmin-antiplasmin+system%3A+structural+and+functional+aspects%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">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/pubmed?term=((A+novel+antithrombotic+protease+from+marine+worm+Sipunculus+nudus%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">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/pubmed?term=((Purification+and+characterization+of+a+novel+fibrinolytic+enzyme+from+culture+supernatant+of+Pleurotus+ostreatus%5BTitle%5D)+AND+%22Journal+of+Microbiology+and+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Starase%3A+A+bi-functional+fibrinolytic+protease+from+hepatic+caeca+of+Asterina+pectinifera+displays+antithrombotic+potential%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">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/pubmed?term=((A+novel+fibrinolytic+protein+From+Pheretima+vulgaris%3A+purification%2C+identification%2C+antithrombotic+evaluation%2C+and+mechanisms+investigation%5BTitle%5D)+AND+%22Frontiers+in+Molecular+Biosciences%22%5BJournal%5D)">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/pubmed?term=((Antioxidant%2C+hypolipidemic+and+hepatic+protective+activities+of+polysaccharides+from+Phascolosoma+esculenta%5BTitle%5D)+AND+%22Marine+Drugs%22%5BJournal%5D)">Antioxidant, hypolipidemic and hepatic protective activities of polysaccharides from Phascolosoma esculenta.</a> Marine Drugs. 18 (3), 158(2020).</li>
<li>Xu, R., Ma, G., Chen, L., Cui, X. Preparation and application of natural fibrinolytic enzyme from peanut worm. , Available from: 
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<li>Li, W., Yuan, M., Wu, Y., Xu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+genes+expressed+differentially+in+female+and+male+gametes+of+Sipunculus+nudus%5BTitle%5D)+AND+%22Aquaculture+Research%22%5BJournal%5D)">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/pubmed?term=((A+simple+method+to+renature+DNA-binding+proteins+separated+by+SDS-polyacrylamide+gel+electrophoresis%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">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/pubmed?term=((DNA+barcoding+reveals+cryptic+diversity+in+the+peanut+worm+Sipunculus+nudus%5BTitle%5D)+AND+%22Molecular+Ecology+Resources%22%5BJournal%5D)">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/pubmed?term=((Plasminogen-plasmin+system.+I.+Purification+and+properties+of+human+plasminogen%5BTitle%5D)+AND+%22The+Journal+of+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Plasminogen-plasmin+system.+II.+Purification+and+properties+of+human+plasmin%5BTitle%5D)+AND+%22The+Journal+of+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Affinity-chromatographic+purification+of+human+%CE%B12-antiplasmin%5BTitle%5D)+AND+%22The+Biochemical+Journal%22%5BJournal%5D)">Affinity-chromatographic purification of human α2-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/pubmed?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%5BTitle%5D)+AND+%22The+Biochemical+Journal%22%5BJournal%5D)">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/pubmed?term=((Purification+of+human+plasma%2Fcellular+fibronectin+and+fibronectin+fragments%5BTitle%5D)+AND+%22Fibrosis%22%5BJournal%5D)">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/pubmed?term=((Expression+analysis+and+purification+of+human+recombinant+tissue+type+plasminogen+activator+%28rt-PA%29+from+transgenic+tobacco+plants%5BTitle%5D)+AND+%22Preparative+Biochemistry+and+Biotechnology%22%5BJournal%5D)">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>
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</ol>

The authors have no conflicts of interest to disclose.

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...

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.

<table><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&#039;,N&#039;-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>&lt;strong&gt;Equipment&lt;/strong&gt;</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>&lt;strong&gt;Consumable Material&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;L PCR Tube (200 &amp;micro;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 &amp;micro;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 &amp;micro;L)</td><td>Axygene</td><td></td><td></td></tr><tr><td>Pipette Tip (200 &amp;micro;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

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&#160;19,200 U/mL,&#160;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.

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

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

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 ...

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Biochemistry

2.1K Views.  Huaqiao University. Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

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

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Cancer Research

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Unveiling Fibrinogenolytic Agents in Peanut Worms with Fibrinogen-PAGE

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus)&#160;in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

Author Spotlight: Time and Cost-Effective Fibrinogen-PAGE for Fibrinogenolytic Studies

Here, we present a fibrinogen-polyacrylamide gel electrophoresis (PAGE) protocol to rapidly separate and display the fibrinogenolytic agents of Sipunculus nudus.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new ...

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)