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，量化细胞培养超自然（第6步）中的蛋白质，以避免超过蛋白质A膜增聚能力。如果检测到蛋白质，则继续。</li>
</ol>2. 准备蛋白质膜增发剂和细胞培养超自然
<ol><li>按照制造商的说明准备膜吸食器。 切勿让空气进入膜沉着器。通过膜吸食器灌注的所有溶液应在室温下使用 0.22 μm 过滤器进行预过滤。 用 0.22μm 过滤的杜尔贝科磷酸盐缓冲盐水 （DPBS） 或选择的缓冲液填充 10 毫升注射器，并释放气泡。香水 DPBS 以去除存储解决方案，并在使用前立即平衡膜增压器。</li>
	<li>使用真空驱动的 0.22μm 无菌过滤装置，在灌注前立即通过膜吸附器过滤细胞培养超自然物。</li>
</ol>3. 加载蛋白质膜增发器
<ol><li>将细胞培养超自然物吸进 Luer Lock 注射器（30 毫升或更大），并释放任何气泡。</li>
	<li>组装材料和设备，如 图1所示。将注射器连接到膜吸食器的入口。将柔性管连接到膜吸食器插座上。如有必要，在注射器和膜吸食器之间放置一个 0.22μm 注射器过滤器。使用烧瓶或瓶子从吸食者处捕获流经（也称为过滤）。</li>
	<li>将注射器泵设置为所需的体积流量，但不超过制造商推荐的流量（例如 10 毫升/分钟）。通过膜增压器将细胞培养超自然体灌注，收集通过烧杯或其他容器的流量。将注射器重新装上所需的超标剂。</li>
	<li>如果需要，使用流动细胞测量、西印、ELISA 等测试蛋白质的流动。基于实验室偏好。通常没有必要反驳流过的流程。</li>
</ol>4. 膜除胶器中的埃卢特蛋白
<ol><li>用 10 毫升 DPBS 清洗膜吸食器，以去除任何非绑定蛋白。</li>
	<li>使用 10-15 毫升的洗涤缓冲器（例如基于胺的洗涤缓冲器（pH 2.8）时，以所需的流速（例如1 毫升/分钟）从膜吸食器中提取的 Elute 蛋白质。在含有中和缓冲器（例如1 M Tris （pH 9.4） 的管中捕获提升，其容量为 10%（1.0-1.5 毫升）。</li>
	<li>或者，将一毫升增量中的蛋白质放入含有 100 μl 中和缓冲液的管中，使用总容量为 10-15 毫升的洗脱缓冲器。使用首选的表征方法测试每个分数中蛋白质的存在。</li>
	<li>根据制造商的说明再生膜沉着器。香水 10 毫升 0.22 μm 过滤 DPBS 或选择缓冲区，然后 10 毫升 0.22 μm 过滤 50 m NaOH 在 1 N NaCl，最后 10 毫升 0.22 μm 过滤 DPBS。在 DPBS 中填充膜吸食器，在 DPBS 中填充 20% 乙醇，以在 4 oC 下进行长期存储。</li>
</ol>5. 浓缩和透析蛋白质
<ol><li>将所有高放（或在第 4.3 步确定的含有蛋白质的浓缩分数）沉积在 10 kDa 分子重量截断离心滤清器单元中。按照制造商的离心指示操作。</li>
	<li>按照制造商的说明，在小容量透析单元（10 kDa 分子重量切断）中对选择的缓冲区进行透析。如果蛋白质沉淀是一个问题，则可能需要在透析材料中添加缓冲区。如果需要，透析材料可以冷藏，直到第 6 步可以执行，但不建议长期存储，不含防腐剂。</li>
</ol>6. 在快速的西方膨胀程序中量化和描述纯化产品
<ol><li>在18-20的减少或不降低条件下，通过 SDS-PAGE 在选择的凝胶上解决纯化蛋白和 Fc 标准。使用迷你凝胶而不是米迪凝胶，以尽量减少运行时间。<ol>
<li>如果尚不为人知，则确定 Fc 标准范围，在加载量（例如 0.1-1 毫克）方面，波段信号的线性变化。使用相同的凝胶、运行条件、转移条件和试剂，用于量化和描述纯化蛋白质。</li>
			<li>加载多种纯化蛋白样本，包括用 DPBS 稀释的样品，以确保纯化蛋白带信号在图像分析中 Fc 标准的线性信号范围内。</li>
		</ol>
</li>
	<li>按照blotter制造商的指示，使用半膨胀的13将蛋白质从凝胶转移到不连续缓冲系统中的聚乙烯氟化物（PVDF）膜。传输时间通常为 5-10 分钟。如有需要，库马西在转移后弄脏凝胶，以验证转移效率21。</li>
	<li>使用真空辅助蛋白检测系统，根据实验室偏好，使用碱性磷酸酶 （AP） 或马萝卜过氧化酶 （HRP） 进行与抗 Fc 或抗 IgG 结合的免疫凝血。免疫印迹时间通常小于1小时。</li>
	<li>使用选择的基板和方法（如 AP基板和增强的化疗）开发免疫体。</li>
	<li>通过图像分析量化纯化蛋白。在来自 Fc 标准的波段信号上执行线性回归。如果 R2>0.90，则使用该线的方程计算其波段信号中的蛋白质数量。如有必要，考虑样品稀释。由于蛋白质的定量是在Fc的基础上进行的，调整蛋白质和Fc之间的分子重量差异值。 如果R2<0.90，则重复整个步骤6。</li>
	<li>使用功能检测或方法验证蛋白质，通过流动细胞测量、西斑、ELISA 等检测 Fc 。基于实验室偏好。</li>
	<li>将蛋白质稀释到所需的浓度和/或在纯化蛋白溶液中加入任何所需的防腐剂或稳定剂，然后再进行长期储存、冷冻或其他补充。</li>
</ol>

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没有什么可透露的。

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。

<table><tbody><tr><td>Protein A membrane adsorber (Sartobind Protein A 2 ml)&amp;nbsp;</td><td>Sartorius-Stedim</td><td>93PRAP06HB-12--A</td><td></td></tr><tr><td>Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td><td>Millipore</td><td>UFC801008</td><td>Use 15 ml volume if needed</td></tr><tr><td>Sterile vacuum filter unit, 0.22 &amp;micro;m, 500 ml</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>10 ml Syringes with Luer Lock tips</td><td>BD</td><td>301604</td><td></td></tr><tr><td>30 ml Syringes with Luer Lock tips</td><td>BD</td><td>309650</td><td></td></tr><tr><td>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><td>Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td><td>Thermo Scientific</td><td>69576</td><td></td></tr><tr><td>Snap i.d. antibody collection tray</td><td>EMD Millipore</td><td>WBAVDABTR</td><td></td></tr><tr><td>Snap i.d. single well blot holder</td><td>EMD Millipore</td><td>WBAVDBH01</td><td></td></tr><tr><td>Elution buffer</td><td>Thermo Scientific</td><td>21004</td><td></td></tr><tr><td>Tris, ultra pure</td><td>Fisher Scientific</td><td>819623</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>7647-14-5</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-100</td><td></td></tr><tr><td>DPBS</td><td>Thermo Scientific</td><td>SH30028.02</td><td></td></tr><tr><td>DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Life Technologies</td><td>14080-055</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Human Fc</td><td>Bethyl Research Laboratories</td><td>P80-104</td><td></td></tr><tr><td>Anti-human Fc-APC</td><td>Jackson Immunoresearch</td><td>109136170</td><td></td></tr><tr><td>Anti-human Fc-AP</td><td>Bio-Rad</td><td>170-5018</td><td></td></tr><tr><td>Alkaline phosphatase substrate</td><td>Promega</td><td>S3841</td><td></td></tr><tr><td>Flow cytometry tubes (5 ml polystyrene or polypropylene)</td><td>BD</td><td>352054</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>55-2226</td><td>A peristaltic pump is also acceptable</td></tr><tr><td>Protein gel electrophoresis system</td><td>Bio-Rad Laboratories</td><td>552BR094876</td><td>Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td></tr><tr><td>Semidry blotter</td><td>Bio-Rad Laboratories</td><td>690BR006163</td><td>Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td></tr><tr><td>SNAP i.d. vacuum-assisted protein detection system</td><td>EMD Millipore</td><td>WBAVDBASE</td><td>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 ...

isolation-characterization-chimeric-human-fc-expressing-proteins

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.1K 次观看.  Ohio University. 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。

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

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

科研

生物化学

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

生物学

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

生物工程

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

细胞调理介质细胞外囊泡的流式细胞学分析

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

DFE-Based Affinity Chromatography: A Technique for Purification of 2F5 Monoclonal Antibodies from a Crude Sample Using High Ionic Strength-Based Elution Conditions

Source: Kn&#246;dler, M. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/59933">Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study</a>. J. Vis. Exp. (2019).
This video describes an affinity chromatography technique for purifying 2F5 monoclonal antibodies using the DsRed-2F5-Epitope or DFE ligands. The purified 2F5 monoclonal antibodies are essential drugs in immunotherapy.

基于 DFE 的亲和层析:一种使用基于高离子强度的洗脱条件从粗样品中纯化 2F5 单克隆抗体的技术

Source: Kn&#246;dler, M. et al., Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case ...