Name: isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow
Uploaded: 2014-01-08T14:15:00
Description: Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

作者要感谢路易斯·德尔加迪略先生（俄亥俄大学化学和生物分子工程系）、马修·威廉斯先生（俄亥俄大学化学和生物分子工程系）和菲利伯托·塞德诺-劳伦特博士（布里格姆妇女医院和哈佛医学院）提供专家技术援助。作者还要感谢2011年冬季CHE 404/BME 504和2012年秋季CHE 4830/BME 5830学生（俄亥俄州大学化学和生物分子工程和生物医学工程系）的技术咨询和讨论。这项工作得到了国家科学基金会（NSF）主要研究仪器赠款CBET-1039869（MMB）、NSF CBET-1106118（MMB）、皮肤病基金会研究补助金A050422（SRB）、国家卫生研究院（NIH）NCI赠款1R15CA161的支持 830-01 （MMB）， NIH 基尔希斯坦-NRSA 博士后奖学金 F32CA14219-01A1 （SRB）， NIH/NCI R01CA173610 （CJD） 和 NIH NCCAM 授予 R01AT004628 （CJD）。

1. 验证蛋白质 A 的亲和力
<ol><li>验证 Fc 表达蛋白（重组融合蛋白或 IgG 抗体， 以下简称"蛋白质"）在使用蛋白A膜吸入器之前，对蛋白质A14-17 具有可接受的亲和力，并测试蛋白质在库存溶液中的存在（例如 ，通过离心在1，000 x g中澄清的细胞培养超纳特，5分钟到颗粒悬浮细胞）。使用流细胞测量、西印、ELISA 等。根据实验室偏好检测 Fc 的蛋白质功能和/或存在。理想情况下?...

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此处描述的协议旨在快速分离和描述表达人类 Fc （Gal-1hFc） 的奇美融合蛋白，同时不会显著影响产品回收或膨胀成本。流线型工作流程的关键组件是用于隔离的蛋白质 A 膜吸附剂，以及用于西式印迹特征的半干转移和真空辅助免疫印迹。
Gal-1hFc 的隔离和特征处理时间从 300 毫升细胞培养超纳特（流线型协议中大约 9 小时，而传统协议中为 20 小时以上）的处理时间的急剧下降?...

使用蛋白质 A 亲和色谱法可以分离来自细胞培养超纳特和稀释裂解细胞溶液的抗体和重组蛋白表达免疫球蛋白 G （IgG） Fc 片段。在基础科学和工程实验室和工业领域，尽管成本高，加工时间长1-3，但富含多孔蛋白A涂层糖、玻璃或聚合物珠子的柱子最常用于亲和色谱。众所周知，亲和色谱是实验室和工业环境中最大的费用和处理瓶颈。因此，为了降低成本和加工时间，在不牺牲从工艺列车1，2，5-7的整体产品回收方面，已经进行了许多改进。抗体和其他Fc表达蛋白的分离特别有希望的是使用蛋白质A膜吸收剂2，5，7-10的亲和色谱。而柱色谱中的 Fc 捕获是扩散限制的（即Fc 表达蛋白必须扩散到内部毛孔并穿过内部毛孔才能到达大多数蛋白质 A），而高压在柱子上下降，膜吸收器中的大量传输由散装对流驱动，从而避免了扩散限制8，9，11，12。此外，膜增压器的压力下降较低，因此与珠子包装柱8，9，11，12相比，进液流速更快。因此，对于实验室规模隔离，膜色谱学预计将减少几个小时的隔离时间，并增加产品捕获量相比，列色谱。此外，还提出了膜吸附器中IgG吸附的数学模型8，9，11，12，从而使最终用户能够预测实验室的性能。
基础科学和工程实验室的另一个瓶颈是Fc表达蛋白的特征。然而，选择适当的方法来完成定性检测，如西方的污点，可以大大减少花费在产品测试上的时间。例如，在不连续的缓冲系统中进行半干旱转移可以在几分钟内完成蛋白质从 SDS-PAGE 凝胶到聚乙烯二氟化物 （PVDF） 膜的电泳传输，而不是在连续缓冲系统13中 1-2 小时的罐（湿）转移。
这里描述了一种快速、廉价和有效的协议，以分离和描述表达人类IgG的Fc片段的奇妙融合蛋白。大多数设备在标准基础生物医学科学、生物学、化学和（生物）化学工程实验室中都随时可用，所有试剂均市售。虽然具有代表性的结果来自粘液加洛汀-1/人类Fc幻想融合蛋白（Gal-1hFc）的分离和特征，但流线型协议可以应用于其他具有Fc片段的分子的分离，如抗体、Fc片段本身或其他Fc融合蛋白。与标准方法相比，我们的改进显著缩短了处理时间（只有 4 小时，但通常为 9 小时，而工作时间为 20 小时以上），同时实现了类似的或改进的产品恢复。

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Protein A membrane adsorber (Sartobind Protein A 2 ml)&#160;</td>
			<td style="width:191px;">Sartorius-Stedim</td>
			<td style="width:147px;">93PRAP06HB-12--A</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td>
			<td style="width:191px;">Millipore</td>
			<td>UFC801008</td>
			<td>Use 15 ml volume if needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sterile vacuum filter unit, 0.22 &#181;m, 500 ml</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">10 ml Syringes with Luer Lock tips</td>
			<td>BD</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">30 ml Syringes with&#160; Luer Lock tips</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tygon lab tubing (1/16 in x 1/8 in x 50 ft)</td>
			<td>Cole Parmer</td>
			<td>WU-95903-16</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td>
			<td>Thermo Scientific</td>
			<td>69576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. antibody collection tray</td>
			<td>EMD Millipore</td>
			<td>WBAVDABTR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. single well blot holder</td>
			<td>EMD Millipore</td>
			<td>WBAVDBH01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Elution buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tris, ultra pure</td>
			<td>Fisher Scientific</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">DPBS</td>
			<td>Thermo Scientific&#160;</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">DPBS with Ca2+/Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Human Fc</td>
			<td>Bethyl Research Laboratories</td>
			<td>&#160;P80-104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-APC</td>
			<td>Jackson Immunoresearch</td>
			<td>109136170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-AP</td>
			<td>Bio-Rad</td>
			<td>170-5018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Alkaline phosphatase substrate</td>
			<td>Promega</td>
			<td style="width:147px;">S3841</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Flow cytometry tubes (5 ml polystyrene or polypropylene)</td>
			<td>BD</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;"></td>
			<td style="width:191px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>55-2226</td>
			<td style="width:173px;">A peristaltic pump is also acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Protein gel electrophoresis system&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>552BR094876</td>
			<td style="width:173px;">Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Semidry blotter&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td style="width:147px;">690BR006163</td>
			<td style="width:173px;">Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:313px;">SNAP i.d. vacuum-assisted protein detection system</td>
			<td style="width:191px;">EMD Millipore</td>
			<td>WBAVDBASE</td>
			<td style="width:173px;">An upgraded model has replaced this specific instrument, but either should work just as well</td>
		</tr>
	</tbody>
</table>

isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow

实验室规模到工业规模净化生物分子从细胞培养超纳特和溶解细胞解决方案可以完成使用亲和力色谱。虽然使用多孔蛋白A的生长珠包装在列中的亲和力色谱可以说是实验室规模隔离抗体和重组蛋白表达IgG片段的最常见方法，但它可能是一个耗时且昂贵的过程。时间和财政拮据在小型基础科学实验室尤其令人生畏，这些实验室必须从稀释溶液中回收数百微克至毫克的蛋白质，但缺乏获得高压液体输送系统和/或具有生物分离专业知识的人员。此外，产品量化和定性也可能在多个工作日过长加工时间，并膨胀费用（消耗品、工资 等）。因此，对于实验室规模隔离和抗体和其他具有 Fc 片段的蛋白质的特征，需要快速、廉价但有效的协议。为此，我们设计了一个协议，由经验有限的技术人员在不到9小时（大约一个工作日）和最快4小时，而不是传统的方法，需要20多工作小时完成。大多数所需的设备在标准生物医学科学、生物化学和（生物）化学工程实验室中随时可用，所有试剂均市售。为了证明这一协议，提出了具有代表性的结果，其中用一种蛋白质A膜增聚剂分离出从细胞培养超母体中融合到人类Fc（Gal-1hFc）的奇美穆林甘胶-1。纯化Gal-1hFc采用西方快速印迹分析程序进行量化，并采用流动细胞学进行特征分析。流线型工作流可用于其他 Fc 表达蛋白（如抗体）和/或更改，以纳入替代定量和定性方法。

简化的Fc表达蛋白分离和特征协议通常用于处理从稀释细胞培养超纳特人融合到人类Fc（Gal-1hFc）的奇美粘膜粘膜加洛汀-1。 图 2 中的流程图说明了协议中每个步骤的工作流程和时间。对于典型的 300 毫升超纳特批次，执行弹性分数的可选流细胞学测试时，总处理时间约为 9 小时。如果省略了所有流细胞分析，由于缺少流量细胞仪或单独的实验室偏好，总处理时间可以短至 4 小时。一般...

Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

与传统的亲和色谱学相比，使用蛋白质A agarose珠状柱，蛋白质A膜吸收剂可以显著加快抗体和其他Fc片段表达蛋白的实验室规模隔离。适当的分析和量化方法可以进一步加快蛋白质加工，使隔离/定性可以在一个工作日内完成，而不是超过20个工作小时。

使用蛋白质膜吸收器和流线型工作流的奇美人 Fc 表达蛋白质的隔离和特征

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

使用光声血流循环黑色素瘤细胞的检测和隔离

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

人体软组织的生物力学特性采用压痕和拉伸试验

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

含有宏观结构的光学组织幻影的制备与表征

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...