本研究由厦门市科技局（3502Z20227197）和福建省科技局（编号：2019J01070，No.2021Y0027）资助。

1. 准备
<ol><li>样品处理<ol><li>仔细解剖新鲜的 裸粒链球菌 （100克）并收集肠道及其内部液体。</li>
		<li>加入 300 mL Tris-HCl 缓冲液（0.02 M，pH 7.4）进行均质化（1，000 rpm，60 s）。</li>
		<li>将匀浆冻融3倍。</li>
		<li>离心样品（10，956× g，0.5小时，4°C）并收集上清液。将样品储存在4°C直至进一步使用。</li>
	</ol></li>
	<li>蛋白质沉淀<ol><li>将上清液与饱和硫酸铵溶液（九体积）混合，让混合物在4°C下静置12小时。 注意：协议可以在此处暂停，稍后继续。</li>
		<li>离心（10，956× g，0.5小时，4°C）得到蛋白质沉淀;将其储存在4°C以备后用。</li>
	</ol></li>
	<li>蛋白质重悬<ol><li>用 30 mL Tris-HCl 缓冲液（0.02 M，pH 7.4）重悬蛋白质沉淀。将此粗蛋白溶液储存在4°C以备后用。</li>
	</ol></li>
</ol>2. 亲和色谱
<ol><li>溶液过滤<ol><li>通过0.22μm滤膜过滤粗蛋白溶液。将此样品储存在4°C以备后用。</li>
	</ol></li>
	<li>色谱柱填料<ol><li>将适量的赖氨酸-琼脂糖基质培养基和精氨酸-琼脂糖基质上样到5 mL空色谱柱中，将精氨酸-琼脂糖基质亲和色谱柱和赖氨酸-琼脂糖基质亲和色谱柱装入5 mL空色谱柱中。 注意：色谱柱可以储存在2-8°C，基质浸入储存缓冲液（20%乙醇）中。</li>
	</ol></li>
	<li>亲和纯化<ol><li>首先用ddH2O平衡亲和色谱柱（1 mL/min，10柱体积），然后使用Tris-HCl缓冲液（0.02 M，pH 8.0，1 mL/min，5柱体积）。</li>
		<li>将蛋白质溶液样品加载到预平衡柱（1 mL/min，1/3柱体积）上。 注意：样品上样量取决于sFE的浓度。</li>
		<li>用Tris-HCl缓冲液（0.02 M，pH 8.0，五柱体积）洗涤色谱柱。</li>
		<li>依次用0.15 M、0.25 M、0.35 M、0.45 M、0.55 M和0.65 M NaCl（1 mL/min，5 个色谱柱体积）洗脱色谱柱。</li>
		<li>寻找应出现在0.15 M NaCl洗脱中的洗脱峰，然后将馏分收集在5 mL管中。</li>
		<li>使用3 kD超离心过滤器（3，944 × g，1.5小时，4°C）浓缩洗脱样品。将此样品储存在-80°C以备后用。</li>
	</ol></li>
</ol>3. 纯度评估
<ol><li>十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶制备<ol><li>根据Laemmli的方法使用5%浓缩凝胶和12%分离凝胶13制备SDS-PAGE凝胶。</li>
	</ol></li>
	<li>电泳<ol><li>将样品（15μL）与5x SDS-PAGE蛋白上样缓冲液（1/4体积）混合，在沸水中加热5分钟，上样到SDS-PAGE凝胶上，并在80 V，60 mA下运行凝胶1.5小时。</li>
	</ol></li>
	<li>分析<ol><li>电泳后，根据制造商的方案用银染试剂盒对凝胶进行染色，并使用化学发光成像系统观察染色凝胶。</li>
		<li>使用以下公式分析目标蛋白质的纯度：目标蛋白质的纯度=目标蛋白质的强度值/总蛋白质的强度值。</li>
	</ol></li>
</ol>4. 纤维蛋白溶解活性评估
<ol><li>纤维蛋白板制备<ol><li>通过将25mg纤维蛋白原与1.25mL生理盐水混合来制备纤维蛋白原溶液。</li>
		<li>通过将100U凝血酶与1.05mL生理盐水完全混合来制备凝血酶溶液。</li>
		<li>通过将 0.5 g 琼脂糖加入 22.5 mL Tris-HCl 缓冲液（0.02 mol/L，pH 7.4）中来制备琼脂糖溶液。混合并在100°C下加热琼脂糖溶液，直到完全溶解。</li>
		<li>将琼脂糖溶液冷却至约50°C并加入纤维蛋白原溶液。然后，立即加入凝血酶溶液，快速混合，并将它们倒入60毫米培养皿中。</li>
	</ol></li>
	<li>装载<ol><li>使用无菌钻孔器在准备好的纤维蛋白板上打孔 3 mm 孔，并用 10 μL 样品填充孔。</li>
	</ol></li>
	<li>分析<ol><li>在37°C孵育18小时后，通过计算其降解区的大小来测量纤维蛋白溶解活性。纤维蛋白溶解活性=（样品的区域大小/尿激酶的区域大小）x 100U.区域大小=直径x直径。 注意：生理盐水缓冲液，粗蛋白（在方案步骤1.3.1中获得的样品）和尿激酶分别用于空白，阴性对照和阳性对照。与尿激酶相比，我们发现纯化的sFE的纤维蛋白溶解活性为~19，200 U / mL。</li>
	</ol></li>
</ol>

通过亲和色谱从 Sipunculus nudus 中纯化纤溶酶

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作者没有利益冲突需要披露。

由于sFE的确切基因序列不可用，目前使用的sFE是从新鲜 的S. nudus14中提取的。此外，文献报道的sFE纯化程序复杂且昂贵，因为它们基于sFE的一些一般特征，如分子量，等电点，离子强度和极性15，16。迄今为止，尚未报道sFE的亲和纯化方案。本研究基于sFE晶体结构知识，成功开发了sFE亲和纯化方案。与报道的纯化方法相比，该亲?...

血栓形成是对公共卫生的主要威胁，尤其是在 Covid-19 全球大流行之后1，2。临床上，许多纤溶酶原激活剂（PAs），如组织型纤溶酶原激活剂（tPA）和尿激酶（UK），已被广泛用作溶栓药物。PA可以将患者的纤溶酶原激活为活性纤溶酶以降解纤维蛋白。因此，它们的溶栓效率受到患者纤溶酶原状态3，4的严重限制。纤维蛋白溶解剂，如金属蛋白酶纤溶酶和丝氨酸纤溶酶，是另一种临床溶栓药物，还包括纤溶酶（FE），如纤溶酶，可直接溶解凝块，但被各种纤溶酶抑制剂迅速灭活5。随后，报道了一种新型的纤维蛋白溶解剂，它不仅可以通过将纤溶酶原激活为纤溶酶，还可以直接降解纤维蛋白6-来自古代花生蠕虫Sipunculus nudus（sFE）的纤维蛋白溶解酶6来溶解血栓6。与传统的溶栓药物相比，这种双功能赋予了sFE其他优势，特别是在纤溶酶原状态异常方面。与其他双功能纤维蛋白溶解剂7，8，9相比，sFE在药物开发方面表现出几个优势，包括安全性，特别是对于口服药物。这是因为海蜥的生物安全性和生物相容性已经确立10.
与从微生物、蚯蚓和蘑菇中分离的其他天然纤维蛋白溶解剂类似，从裸蚯蚓中纯化sFE非常复杂，包括组织匀浆、硫酸铵沉淀、脱盐、阴离子交换色谱、疏水相互作用色谱和分子筛等多个阶段10，11，12.这样的净化系统不仅要依靠熟练的技能和昂贵的材料，还需要几天的时间才能完成整个过程。因此，一个简单的sFE纯化方案对sFE的进一步发展具有重要意义。幸运的是，两个sFE晶体（PDB：8HZP;PDB：8HZO）已成功获得（见补充文件1和补充文件2）。通过结构分析和分子对接实验，我们发现sFE的催化核心可以特异性地与含有精氨酸或赖氨酸残基的靶标结合。
本文首次提出了一种基于sFE晶体结构的亲和纯化系统。通过遵循该方案，可以在单个亲和纯化阶段从粗提取物中纯化高纯度和高活性sFE。这里开发的方案不仅对sFE的大规模制备很重要，而且可用于纯化其他纤维蛋白溶解剂。

<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

海蕊纤溶酶（sFE）是一种新型纤溶剂，既能将纤溶酶原激活成 纤 溶酶，又能直接降解纤维蛋白，与传统溶栓剂相比具有很大的优势。然而，由于缺乏结构信息，所有sFE的纯化程序都基于多步色谱纯化，过于复杂和昂贵。本文首次基于sFE的晶体结构开发了sFE亲和纯化方案;它包括粗样品和赖氨酸/精氨酸-琼脂糖基质亲和色谱柱的制备、亲和纯化和纯化 sFE 的表征。按照该方案，一批sFE可以在1天内纯化。此外，纯化的sFE的纯度和活性分别增加到92%和19，200 U/mL。因此，这是一种简单、廉价且高效的sFE纯化方法。该方案的开发对于sFE和其他类似药物的进一步利用具有重要意义。

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.

作者聚焦：从 Sipunculus nudus 中亲和纯化纤溶酶  

从海蛾中亲和纯化纤维蛋白溶解酶

Our research is mainly about the purification of fibrinolytic enzyme. This work tries to establish a simple purification method for fibrinolytic enzyme. In this work, we report an arginine or lysine best-affinity purification system of fibrinolytic enzyme for the first time.Compared with the conventional purification methods, this is a simple, inexpensive, and efficient approach for the purification of fibrinolytic enzymes from Sipunculus nudus. The development of this protocol is of greater significance for the further utilization of fibrinolytic enzymes from Sipunculus nudus and other similar agents.

按照该方案，提取粗组织裂解物，建立精氨酸-琼脂糖基质和赖氨酸-琼脂糖基质亲和色谱柱，得到纯化的sFE，分别用SDS-PAGE和纤维蛋白板测定纯化sFE的纯度和纤维蛋白溶解活性。
离心后，收集的上清液为透明的棕褐色粘稠液体。当该上清液与饱和硫酸铵溶液（九体积）混合时开始沉淀。静置12 h后，在管底形成重沉淀。当用Tris-HCl缓冲液重悬时，蛋白质沉淀迅速消失并溶解在缓?...

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

在这里，我们提出了一种来自 海蜥蜴 纤维蛋白溶解酶的亲和纯化方法，该方法简单、廉价且高效。

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

我们的研究主要是纤维蛋白溶解酶的纯化。这项工作试图建立一种简单的纤维蛋白溶解酶纯化方法。在这项工作中，我们首次报道了纤维蛋白溶解酶的精氨酸或赖氨酸最佳亲和力纯化系统。与传统的纯化方法相比，这是一种简单、廉价且有效的纯化海藻纤维蛋白溶解酶的方法。该协议的开发对于进一步利用来自海藻的纤溶酶和其他类似试剂具有更大的意义。

affinity-purification-of-a-fibrinolytic-enzyme-from-sipunculus-nudus

Research

JoVE Journal

Biochemistry

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

1.2K 次观看.  Huaqiao University. 我们的研究主要是纤维蛋白溶解酶的纯化。这项工作试图建立一种简单的纤维蛋白溶解酶纯化方法。在这项工作中，我们首次报道了纤维蛋白溶解酶的精氨酸或赖氨酸最佳亲和力纯化系统。与传统的纯化方法相比，这是一种简单、廉价且有效的纯化海藻纤维蛋白溶解酶的方法。该协议的开发对于进一步利用来自海藻的纤溶酶和其他类似试剂具有更大的意义。