Name: affinity purification of a fibrinolytic enzyme from sipunculus nudus
Uploaded: 2023-06-02T12:10:00.000+00:00
Duration: 6 min 45 s
Description: Watch this Scientific Journal Video about Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus at JoVE.com

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><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

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。

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

作者聚焦：从 Sipunculus nudus 中亲和纯化纤溶酶  

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。

从海蛾中亲和纯化纤维蛋白溶解酶

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

2.0K 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。

视频: 作者聚焦：从 Sipunculus nudus 中亲和纯化纤溶酶  

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.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

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

科研

生物化学

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.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

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.

从真核细胞蛋白质复合体串联亲和纯化

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.

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

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.

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

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.

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

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

癌症研究

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.

从猪肾和小鼠肝脏中提取和纯化FAHD1蛋白

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

<meta charset="utf8"></head><body>使用 Fibrinogen-PAGE 揭示花生蠕虫中的纤维蛋白原溶解剂

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.

<meta charset="utf8"></head><body>作者聚焦：用于纤维蛋白原溶解研究的时间和成本效益的纤维蛋白原-PAGE

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

从海蛾中亲和纯化纤维蛋白溶解酶

副本

摘要

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此视频中的章节

从外膜囊泡内蛋白质导演包装

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

从真核细胞蛋白质复合体串联亲和纯化

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

从猪肾和小鼠肝脏中提取和纯化FAHD1蛋白

通过纤维蛋白原-聚丙烯酰胺凝胶电泳快速分离和展示 Sipunculus nudus 中的活性纤维蛋白原溶解剂

通过纤维蛋白原-聚丙烯酰胺凝胶电泳快速分离和展示 Sipunculus nudus 中的活性纤维蛋白原溶解剂

通过纤维蛋白原-聚丙烯酰胺凝胶电泳快速分离和展示 Sipunculus nudus 中的活性纤维蛋白原溶解剂